Compositions and methods for performing magnetibuoyant separations

ABSTRACT

Processes and compositions are provided for performing magnetibuoyant separations of different biomolecules (e.g., cells, organelles, etc.) in a biological sample, as well as compositions and kits for performing such methods. Compositions containing the separated biomolecules, and methods for using the same for in-vitro and in-vivo biomedical applications, are also provided. The magnetibuoyant methods of the invention employ targeted magnetic particles, preferably targeted nanomagnetic particles, and targeted buoyant particles such as buoyant microparticles and microbubbles. Among the benefits of the invention is the ability to combine targeted magnetic particles with differentially targeted buoyant particles to achieve separation of two or more specifically cell targeted populations during the same work flow.

RELATED APPLICATION

This application claims the benefit of and priority to U.S. provisionalpatent application Ser. No. 62/330,112 (attorney docket numberBLD-0015-PV), filed 30 Apr. 2016, also entitled, “Compositions andMethods for Performing Magnetibuoyant Separations”, the contents ofwhich are hereby incorporated by reference in their entirety for any andall purposes.

BACKGROUND OF THE INVENTION

Magnetic particle-based technologies for the separation and isolation ofcells, nucleic acids, proteins, and other biomolecules have becomeestablished and improved over the past several decades. Magneticparticles are typically conjugated with specific targeting moieties suchas antibodies or nucleic acids, allowing the particles to bind to thetarget molecules found in complex mixtures such as cell populations orprotein and nucleic acid mixtures. The magnetic particles bound to thetarget biological material can then be separated from the mixture usingmagnetic field devices, providing a purification or enrichment methodfor the target. Such magnetic particle-based biological target isolationapproaches have been used to isolate or enrich eukaryotic cells bearingtarget antigens, bacterial species, nucleic acids, and proteins forresearch and therapeutic uses. They have also been used in clinicaltesting applications such as serving as solid supports for immunoassaysor radioimmunoassays (RIA).

Methods for preparing magnetic particles for such applications aretypically of two general types. One general method involves dispersingthe magnetic particles evenly within a polymeric matrix duringpreparation of the polymeric particles, constructing a magnetic materialshell around a polymeric particle core, or introducing magnetic materialinto pre-existing pores within the polymer particles. Examples of theformer method can be found, for example, in U.S. Pat. No. 4,358,388, andof the second method in U.S. Pat. Nos. 5,320,944 and 5,091,206. Thelatter method is exemplified in, for example, U.S. Pat. Nos. 5,648,124and 4,654,267. All of these methods result in magnetic particles ofgreater than 0.3 um (micrometer) in size.

The second general method for preparing magnetic particles forbiomaterial applications involves creating bare magnetic materialparticles first that serve as the core of a larger particle created byconstructing a shell around the first magnetic material core. One formof primary coating has been a silane coat, but other coatings have alsobeen described. For example, U.S. Pat. No. 3,933,997 describes the useof a silane coupling agent that coats magnetic particles and directlyconjugates to specific antibodies. This material was reportedly intendedfor use in RIA methods. U.S. Pat. No. 4,554,088 describes constructionof a metal or iron oxide particle core that is coated by a polymericsilane to which bioaffinity molecules such as antibodies are directlycoupled. U.S. Pat. No. 4,695,392, a division of the aforementioned '088patent, further defines the silane coat to which bioaffinity moleculesare directly attached as having two discrete functionalities—the firstto adsorptively or covalently couple to the metal oxide core particleand the second to covalently couple to bioaffinity organic molecules. Inboth patents the size of particles is defined as ranging from 0.1 um to1.5 um. U.S. patent application publication no. 2007/0026435, nowabandoned, discloses a hydroxysilane, preferablyhydroxyalkyltrialkoxysilane, primary coating on a magnetic particlecore. In this application the particle sizes ranged from 0.1 um to 100um, and the particles were specified for use in isolation of specificnucleic acids from mixtures. The magnetic particles disclosed in boththe '392 patent and the 2007/0026435 publication produce highlyaggregated magnetic particles upwards of 1 um in diameter when strictlyadhering to the cited examples contained therein. U.S. Pat. No.7,169,618 discloses preparation of magnetic particles of a size rangefrom 0.07 um to 0.45 um that are first coated with an organosilane thatis then conjugated with a polysaccharide material via a pendantfunctional group on the organosilane. U.S. patent applicationpublication no. 2010/0012880 discloses a magnetic particle having amagnetic material core with a primary hydrophobic protective layer overwhich is layered a hydrophilic alkylsilane coating. Such particles aredisclosed as being from 0.2 um to 0.4 um in diameter.

Distinct from silane coatings that also serve as the coupling reagent tobioaffinity molecules, non-silane primary coatings on core magneticparticles have also been reported. These include polyglutaraldehyde(see, e.g., U.S. Pat. No. 4,267,234), acrylamide, n-butylacrylate, orN,N′-methylenebisacrylamide (see, e.g., U.S. Pat. No. 4,454,234),polyacrolein (see, e.g., U.S. Pat. No. 4,783,336), polyvinyl alcohol(see, e.g., U.S. Pat. No. 6,204,033), natural polymers like dextran(see, e.g., U.S. Pat. No. 4,452,773), and bovine serum albumin (see,e.g., U.S. Pat. No. 4,795,698). All of these magnetic particle primarycoatings reportedly serve as substrates to which additional biomoleculessuch as antibodies or nucleic acids may be conjugated. With all of thesemethods, the shapes and sizes of the resultant bioaffinity magneticparticle products are not easily controlled, the size range of theparticle products are relatively broad, the diameters are typicallygreater than 0.5 um, and the product particles tend to easily adhere toone another forming particle clumps.

More recently materials have become available for the successfulseparation and isolation of cells using floatation of the desired cells.This is conceptually similar to the use of targeting moieties conjugatedto magnetic particles, except in this case the particles are buoyant ina biocompatible isolation buffer. The buoyant particles can lift thetargeted cell or biomolecule away from unwanted components to thesurface of an isolation vessel where the buoyant particles and cells orbiomolecules can be harvested.

For cell and biomolecule enrichment and separation, all availableparticle-based systems are restricted to a single direction and theenrichment and/or isolation of a single population. For example, withmagnetic particles, including magnetic nanoparticles, positive selectionof a cell population occurs by moving the desired cell toward a magneticpole if sufficient magnetic nanoparticles have bound to the cell via thetargeting moiety. The magnetic particles may be conjugated with one ormany targeting vectors, or a population of particles may contain manysubsets of particles conjugated with different targeting moieties. Inthe latter case the magnetic particle mixture is used to move severaldifferent cell or biomolecule types toward the magnetic pole. In manyinstances enriching and isolating cells or particles based on movementin a single direction is sufficient to isolate a desired population.However, there are many cases where it would be highly advantageous tobe able to rapidly and easily move at least two different populations indifferent directions so that two or more different cell or biomoleculepopulations may be enriched and isolated from a starting complexmixture, or so that purification of a single population is improvedthrough the rapid removal of a difficult contaminating population. Thus,there is a need for methods, materials and kits that allow for therapid, simple and affordable movement of cells and biomoleculescontained within complex mixtures in at least two directions within asingle work flow.

The present invention satisfies the need for simple and rapidbi-directional separation of two or more cell or biomolecule populationsfrom complex mixtures using stable magnetic particles (preferably,magnetic nanoparticles) conjugated with specific targeting moieties andany of several forms of buoyant microparticles (e.g., microbubbles)conjugated with additional targeting moieties.

SUMMARY OF THE INVENTION

The object of this invention is to provide simple and rapidbi-directional separation of two or more cell or biomolecule populationsfrom complex mixtures using stable magnetic particles (preferably,magnetic nanoparticles) conjugated with specific targeting moieties andany of several forms of buoyant particles (e.g., microbubbles)conjugated with additional targeting moieties.

Thus, one aspect of the invention concerns methods of separating atleast one target biomolecule species from a biological sample. Suchmagnetibuoyant separation methods comprise forming a reaction mixtureand contacting a biological sample known or suspected to contain firstand second biomolecule species of interest with a targeted magneticparticle species, optionally a targeted nanomagnetic particle species,that targets the first biomolecule species of interest to form firsttarget biomolecule/magnetic particle complexes and a targeted buoyantparticle species, optionally a targeted buoyant microparticle,optionally a microbubble, species that targets the second biomoleculespecies of interest to form second target biomolecule/buoyant particlecomplexes. A magnetic field to isolate the first biomolecule/magneticparticle complexes from the reaction mixture and buoyancy/floatationproperties are used to separate the second target biomolecule/buoyantparticle complexes from the reaction mixture. The magnetic andbuoyancy/floatation separations can be performed in series or inparallel, and the order of separations can vary. In some preferredembodiments, the targeted magnetic particles and/or the targeted buoyantparticles each independently further comprise a detectable label. Insome preferred embodiments, the targeting moiety of the targetedmagnetic particle species and the targeted buoyant particle species aredifferent and each is independently selected from the group consistingof an antibody, an antigen-binding antibody fragment, a recombinantantibody, a cell surface receptor, a ligand-binding extracellular domainof a cell surface receptor, an aptamer, a nucleic acid, avidin,streptavidin, and biotin. In some preferred embodiments, the targetedbuoyant particle species comprises targeted microparticles, optionallytargeted microbubbles. In some preferred embodiments, the firstbiomolecule species is a cell-surface antigen of a cell type useful forcell therapy, optionally human cell therapy.

In preferred embodiments of this aspect, the targeted magnetic particlespecies is a targeted nanomagnetic particle species that comprises amagnetic core particle, a glass layer encapsulating the magnetic coreparticle, a protein/polymer composite layer bound to the glass layer,and a targeting moiety that targets the first biomolecule species ofinterest and comprises one member of a bioaffinity ligand pair bound tothe protein/polymer composite layer. In some preferred embodiments, themolecules of the targeted nanomagnetic particle species have a diameterranging from about 5 nm to about 500 nm, preferably from about 30 nm toabout 300 nm. In some preferred embodiments, the magnetic core particlesof the targeted nanomagnetic particle species comprise magnetite (Fe₃O₄)crystals, optionally wherein the magnetite crystals have a diameterranging from about 5 nm to about 300 nm. In some preferred embodiments,the glass layer of the targeted nanomagnetic particle species is asilane layer formed from organofunctional alkoxysilane molecules,optionally organofunctional alkoxysilane molecules that comprise acouplable end group, optionally a couplable end group selected from thegroup consisting of an amino, sulphydryl, carboxyl, and hydroxyl endgroup. In some preferred embodiments, the protein/polymer compositelayer of the targeted nanomagnetic particle species is covalently boundto the glass layer, optionally wherein the protein/polymer compositelayer is comprised of serum albumin, optionally bovine or human serumalbumin, dextran or casein and wherein optionally the protein/polymercomposite layer is permanently bound by heating the composition fromabout 45° C. to about 85° C. In some preferred embodiments, thetargeting moiety of the targeted nanomagnetic particle species isselected from the group consisting of an antibody, an antigen-bindingantibody fragment, a recombinant antibody, a cell surface receptor, aligand-binding extracellular domain of a cell surface receptor, anaptamer, a nucleic acid, avidin, streptavidin, and biotin. In somepreferred embodiments, the magnetic core particles of the targetednanomagnetic particle species comprise a ferrous oxide, optionally Fe₃O₄or Fe₂O₃; a chromium oxide, optionally CrO₃; or a stable metal oxidethat comprises a substituted metal ion selected from the groupconsisting of Mn, Co, Ni, Zn, Gd, and Dy.

The nanomagnetic particles so produced have three layers of coatingsaround the core nano-sized magnetic particles, namely a silane or glasslayer, a protein/polymer layer, and finally an outermost layer that iscomprised of targeting moieties, which are one member of a bioaffinityligand pair, such as an antibody for targeting an antigen of interest, acell surface receptor or receptor fragment, etc. The targeting moiety orbioaffinity ligand (which may be, for example, an antibody orantigen-binding antibody fragment, streptavidin, peptide, nucleic acidpolymer, or other receptor or ligand of interest) is preferablycovalently conjugated to the ample functional groups present on theprotein/polymer layer. In preferred embodiments, the glass layer is asilane layer formed from organofunctional alkoxysilane molecules,optionally organofunctional alkoxysilane molecules that comprise acouplable end group, optionally a couplable end group selected from thegroup consisting of an amino, sulphydryl, carboxyl, and hydroxyl end orreactive group. The end group may be protected or unprotected; ifprotected, a deprotection step is preferably used prior to coupling ofthe protein/polymer composite layer. In preferred embodiments, theprotein/polymer composite layer is covalently bound to the glass layer.Preferably, the protein/polymer composite layer is comprised of serumalbumin, e.g., bovine or human serum albumin, dextran, or casein. Insome embodiments, the protein/polymer composite layer is permanentlybound by heating the composition from about 45° C. to about 85° C. Thetargeting moiety or bioaffinity ligand (i.e., one member of a highaffinity binding pair) is then conjugated, preferably covalently, to theprotein/polymer layer. Preferred targeting moieties include antibodies(preferably monoclonal antibodies), antigen-binding antibody fragments(e.g., Fab fragments), cell surface receptors, ligand-bindingextracellular domains of cell surface receptors, nucleic acids(including nucleic acid-based aptamers), avidin, streptavidin, biotin,and pharmaceutical compounds for purposes of targeted drug delivery.

The targeted nanomagnetic particles of the invention behave as stablecolloids when combined in a reaction mixture with complex liquids, forexample, mammalian whole blood or a fraction of mammalian whole blood.Moreover, targeted nanomagnetic particles of the invention preferablyexhibit no significant or deleterious change in magnetic, bioaffinity,and/or particle size and targeting properties during storage over longperiods, e.g., 1 year to 5 years. Preferred sources of biologicalsamples are those obtained from mammals, including humans, as well asfrom companion animals (e.g., cats and dogs) or those of commercialsignificance (e.g., cattle; fowl such as chickens, turkeys, and ducks;goats; horses, pigs, sheep, etc.).

Compositions comprising the targeted magnetic (nano)particles andtargeted buoyant microparticles of the invention can be formulated inany suitable manner, including dry, readily dispersible formulations(e.g., lyophilized formulations) or liquid compositions. Afterpreparation, such compositions are typically dispensed in desiredquantities (e.g., in an amount suitable for performing a single magneticseparation, or alternatively, multiple separations) into suitablecontainers that are then often packaged into kits for subsequentdistribution and use. Kits according to the invention preferably includeinstructions for use of the reagents in the kit, including use of thetargeted (nano)magnetic particles and targeted buoyant microparticles ofthe invention to perform one or more desired magnetic separations. Insome embodiments, such kits may include a plurality of targeted magneticparticle species (all, some, or none of which may include nanomagneticparticles) and targeted buoyant microparticles (all, some, or none ofwhich may include targeted microbubbles), wherein each targeted(nano)magnetic particle species and targeted buoyant microparticlespecies comprises a different targeting moiety species. Preferably, inkits that contain a plurality of different targeted magnetic particlespecies and targeted buoyant particle species, each species ispreferably packaged in a separate container in the kit. Such kits mayalso include other reagents, equipment, and supplies needed forperforming magnetic and buoyant separations of one or more particularbiomolecule species from a reaction mixture prepared from a biologicalsample.

Thus, this invention relates to the combined use of magnetic separationand buoyant separation to enrich and separate target biomolecules, forexample, cells, organelles, exosomes, oncosomes, and other biologicalmaterials to be isolated from complex mixtures such as biologicalsamples.

Another aspect of the invention relates to using the magnetibuoyantseparation methods of the invention to prepare enriched cellpopulations, wherein the cells of the enriched cell population expressthe first biomolecule species as a cell-surface antigen.

A related aspect concerns isolated, enriched cell populations producedusing a magnetibuoyant separation method according to the invention.

Yet a further related aspect relates to methods of administering anenriched cell population to a subject, for example, a human. Suchmethods comprise administering to a subject an isolated, enriched cellpopulation of the invention, for example, a cell population enriched forstem cells that express the first biomolecule species as a cell-surfaceantigen.

Another aspect of the invention relates to kits for performingmagnetibuoyant separations according to the invention. Such kitstypically include at least one composition that comprises a targetedmagnetic particle species, optionally a targeted nanomagnetic particlespecies, that targets a first biomolecule species of interest, at leastone composition that comprises a targeted buoyant particle species,optionally a targeted buoyant microparticle species, optionally atargeted buoyant microbubble species, that targets a second biomoleculespecies of interest, which composition targets a different biomoleculespecies as compared to that targeted by the composition comprising thetargeted magnetic particle species, and instructions for performing amagnetibuoyant separation using the targeted magnetic particle speciesand targeted buoyant particle species. In some embodiments, the kitincludes a plurality of targeted magnetic particle species, wherein thebiomolecule species of interest targeted by each targeted magneticparticle species is different from other biomolecule species of interesttargeted by other targeted magnetic particle species and the targetedbuoyant particle species in the kit, wherein the different targetedmagnetic particle species are in the same or different compositions inthe kit. In some embodiments, the kit includes a plurality of targetedbuoyant particle species, wherein the biomolecule species of interesttargeted by each targeted buoyant particle species is different fromother biomolecule species of interest targeted by other targetedmagnetic particle species and targeted buoyant particle species in thekit, wherein the different targeted buoyant particle species are in thesame or different compositions in the kit. In other embodiments, the kitincludes a plurality of targeted buoyant particle species and aplurality of targeted magnetic particle species.

Other aspects of the invention concern methods for the magnetibuoyantisolation, separation, concentration, and purification of desired, ortarget, biomolecules, e.g., cells, organelles, etc. According to variousembodiments of these aspects, such methods comprise the steps ofproviding in solution targeted magnetic particles, particularly targetednanomagnetic particles and targeted buoyant particles, particularlytargeted buoyant microparticles (especially microbubbles), each coatedwith different targeting moiety species, contacting the targetedmagnetic and buoyant particles with a plurality of biomolecular species(e.g., different types of cells), such as may be present in a biologicalsample, that interact with their targeting moieties in solution to formfirst biomolecule/magnetic particle complexes and secondbiomolecule/buoyant particle complexes, and separating the complexes soformed from the solution. In preferred embodiments, the firstbiomolecule/magnetic particle complexes are magnetically separated fromthe solution and the second biomolecule/buoyant particle complexes areallowed to rise, or float, to the top of the solution and are thenremoved, thereby separating the second biomolecule/buoyant particlecomplexes from the solution. In this manner, all manner of biomoleculespecies can be magnetibuoyantly isolated, separated, concentrated, orpurified. Representative examples of biomolecular species include cells,cellular components (liposomes, endoplasmic reticulum, etc.),subcellular organelles (mitochondria, etc.), and components ofsubcellular organelles, as well as complexes thereof, as well asproteins (antigen, antibodies, ligands, receptors, hormones), nucleicacids (RNA, DNA nucleotide analogs, mixtures thereof, etc.),lipoproteins, fats, triglycerides, sugars, and carbohydrates. Isolation,separation, concentration, and purification can re by enrichment,depletion, or a combination of enrichment or depletion steps. Also, themagnetic and buoyant separations may be performed sequentially or inparallel. When sequential, in some embodiments the magneticseparation(s) may be performed before the buoyant separation(s), whilein other embodiments the buoyant separation(s) are performed before themagnetic separation(s).

As will be appreciated, the targeted buoyant particles (e.g., targetedmicrobubbles) of the invention bind to a desired target biomoleculespecies, such as cells, viruses, analytes, or other biomolecules byvirtue of the targeting moiety species coupled to the buoyant particlesand then rise to the surface of the solution, thus separating themselvesfrom the non-target species in the reaction mixture. In certainembodiments, where the separation time is important, the reactionmixture containing the target molecule/buoyant particle complexes may becentrifuged or subjected to a bubble trap to further effect theseparation more rapidly. The targeted buoyant particle component of theinstant magnetibuoyant separation methods also offers an additionaladvantage in that in the normal force of gravity and the buoyant forceof the buoyant particles are in different directions, thus resulting ina significant reduction in non-specific binding and entrapment ofbiomolecules that typically sink toward the bottom of the reactionvessel during separation. Separation can be enhanced with unboundbiomolecules being forced away from the targeted buoyant particles in alow centrifugal field, as with a modest centrifugal speed, underconditions that do not adversely affect the targeted buoyant particles.

In embodiments of the invention that utilize protein-based (e.g.,albumin-based) targeted microbubbles, such microbubbles have the usefulproperty of being able to be easily destroyed or disrupted and be madeto visually disappear by applying pressure or vacuum to the solution, orby adding a small amount of a detergent or surfactant. This can beparticularly useful where it is desirable to isolate the targetedbiomolecule species devoid of the capturing microbubble. For example, itmay be desirable to characterize the phenotype of a buoyantly separatedcell or to free the isolated cell for further analysis or propagation.Such approaches can avoid potentially damaging reagents, such asenzymes, harsh chemicals, and pH extremes. In other embodiments, ifdesired the targeted biomolecule species of interest may be releasedfrom the microbubble by enzymatic or chemical approaches.

These and other aspects, objects, and embodiments of the presentinvention, which are not limited to or by the information in thisSummary, are provided below, including in the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an acid dissolution study performed onnon-silanized silanized and silanized nanomagnetic particles of theinvention pre- and post-sonication. The plot shows the percentage ofiron dissolved after a 15 min. exposure to 4 M HCl.

FIG. 2 shows the magnetic separation efficiency of various nanomagneticparticles made according to this invention.

FIG. 3 shows the magnetic separation efficiency of anantibody-conjugated commercially available magnetic nanoparticleproduct.

FIG. 4 shows the purity of CD4 positive cells that were negativelyselected using Streptavidin-conjugated nanomagnetic particles andappropriate biotinylated antibodies that were stored at varioustemperatures and then tested for cell separation performance over thecourse of two months.

FIG. 5 shows the CD4 positive cell yield of Streptavidin-conjugatednanomagnetic particles that were stored at various temperatures andtested for cell separation performance as in FIG. 4 over the course oftwo months.

FIG. 6 shows the purity of rat anti-mouse CD19 antibody-conjugatednanomagnetic particles that were stored at various temperatures andtested for CD19 positive cell separation performance over the course oftwo months.

FIG. 7 shows the yield of rat anti-mouse CD19 antibody-conjugatednanomagnetic particles that were stored at various temperatures andtested for CD19 positive cell separation performance over the course oftwo months.

FIG. 8 is a plot showing the particle size distributions of variousconventional, commercially available magnetic particles compared tothose of the invention produced in accordance with Example 1, below.Measurements were made using dynamic light scattering and the percentageof particles in various ‘size-bins’ was plotted as a function of actualparticle size.

FIGS. 9A-9F, each of which is a representation of a transmissionelectron micrograph. The micrographs in FIGS. 9A, 9C, and 9E representcells magnetically selected by HGMS using commercially HGMS compatiblemagnetic particles, while FIGS. 9B, 9D, and 9F represent cellsmagnetically selected by HGMS using targeted nanomagnetic particles ofthe present invention.

FIG. 10 shows a plot of relative fluorescence units (RFU) versusconcentration of anti-mouse CD3 antibody used for coating microwells todrive the cells to proliferate. RFU is an index of the relative numberof cells in each condition.

FIG. 11 illustrates the general scheme for “magnetibuoyant” separationmethods of the invention for the rapid enrichment and isolation of twoseparate cell populations (cell populations labeled “A” and “B”) from acomplex mixture (cell populations labeled “A”, “B”, “C”, and “D”). Cellsof cell population A are isolated by floatation with targetedmicrobubbles (a type of buoyant particle) while cells B are isolated bytargeted magnetic particles. N and S represent North and South poles ofa magnetic separator device.

FIG. 12 illustrates the general scheme for “magnetibuoyant” separationmethods of the invention for the rapid enrichment and isolation of threeseparate cell populations (cell populations labeled “A”, cells “B”, andcells “C”) from a complex mixture (cell populations labeled “A”, “B”,“C”, and “D”). Cell populations A and Cells B are isolated as in FIG.11, while cell populations C plus D are sedimented and isolated aftercell population A has been removed from the same tube. N and S representNorth and South poles of a magnetic separator device.

FIG. 13 illustrates the principle of using targeted buoyant particlessuch as targeted microbubbles to enrich or isolate a wanted populationof cells (CD19⁺ lymphocytes) from a complex cell mixture (mousesplenocytes). CD19 antibody-conjugated microbubbles are mixed (a) withthe complex mixture containing cells if types “A”, “B”, and “C”. CD19⁺cells (A) are floated to the surface by microbubble-conjugated antiCD19antibodies (b). CD19⁺ cells are harvested from the surface andtransferred to another vessel (c) for further use.

FIG. 14A illustrates the principle of using targeted buoyant particlessuch as targeted microbubbles to enrich or isolate a wanted populationof cells (hematopoietic stem cells (labeled “D”)) from a complex mixtureof mostly unwanted cells (mouse bone marrow cell types (labeled “A”“D”)). In this example, cells of type “C” are an acceptable contaminantof the enriched population. The complex cell mixture is incubated withmicrobubble-conjugated antibodies that recognize most of the unwantedcells (a) that are floated to the surface (b), leaving the wanted cells(“D”) and the acceptable contaminant cell (“C”). The unwanted cells (Aand B) are removed from the surface to leave the wanted (D) andacceptable contaminant cells (C) for further use. FIGS. 14B and 14C arecytograms. The lower panel (FIG. 14C) shows flow cytometric analysisdata (see Example 13, below) illustrating enrichment of the wantedhematopoietic stem cells from a starting frequency of 0.52% (Left, Q2)to 3.77% (Right, Q2).

FIG. 15A illustrates one example of simultaneous application of the“magnetibuoyant” methods of the invention for rapid enrichment andseparation of two discrete populations of cells. FIG. 15A depicts thatthe complex mixture (mouse spleen mononuclear cells) is simultaneouslyexposed to both anti CD4 antibody-conjugated microbubbles (a type oftargeted buoyant particle) and anti CD19 antibody-conjugated magneticnanoparticles (a type of targeted magnetic particle) (a). The CD4⁺ cells(B) are floated to the surface while the CD19⁺ cells (A) are drawn tothe magnetic device at the vessel walls. The unwanted CD4⁻/CD19⁻ cells(C) sediment (b). Two desired cell populations (A and B) are separatelyharvested and transferred to individual vessels for further use (c). Theunwanted CD4⁻/CD19⁻ cells (C) are discarded. N and S represent North andSouth poles of a magnetic separator device. FIGS. 15B-15D are cytograms.The lower panel (FIG. 15D) shows flow cytometric analysis data (seeExample 14, below) illustrating enrichment of the CD19⁻/CD4⁻ cells (A)from a starting frequency of 50.5% (Control, Q3) to 98.3% (AfterSeparation PF, Q3), and the CD4/CD19 phenotype of the residual unwantedcell mixture (C, After Separation (NF)).

FIG. 16 illustrates one example of sequential application of the“magnetibuoyant” methods of the invention for improved and rapidenrichment and separation of a discrete population of cells. The complexmixture (human PBMC) is simultaneously exposed to both anti CD14antibody-conjugated microbubbles and anti CD4 antibody-conjugatedmagnetic nanoparticles (a). The CD14⁺ cells (B) are first floated to thesurface, harvested, and discarded (b). The CD4⁺ cells (A) are then drawnto the magnetic device at the vessel walls (c). The unwanted CD14⁻/CD4⁻cells (C) sediment and are discarded (c). The desired and highlyenriched CD4⁺ cell population (A) is harvested and transferred to aseparate vessel for further use (d). N and S represent North and Southpoles of a magnetic separator device.

FIGS. 17A-17F illustrate flow cytometric analysis (see Example 15,below) of a highly enriched CD4⁺/CD14⁻ obtained from complex human PBMCsas described in FIG. 16. All cytogram axes show CD14 (ordinate) by CD4(abscissa). FIG. 17A, Control: freshly prepared PBMCs, no purificationsteps. FIG. 17B, After CD14 depletion: remaining PBMC population afterremoval of CD14⁺ cells using microbubbles as in step (b) of FIG. 16.FIG. 17C: Without CD14 depletion row, CD4 positive fraction: CD4positively selected fraction obtained using magnetic particles withoutCD14 pre-removal. FIG. 17D: Without CD14 depletion row, CD4 negative:Negative fraction remaining after CD4 positive selection step usingmagnetic particles without CD14 pre-removal. FIG. 17E: With CD14depletion row, CD4 positive fraction: CD4 positively selected fractionobtained after pre-removal of CD14⁺ cells. FIG. 17F: With CD14 depletionrow, CD4 negative fraction: negative fraction remaining after positiveselection removal of CD4⁺ cells and after CD14 pre-removal step.

DETAILED DESCRIPTION

As those in the art will appreciate, the following detailed descriptiondescribes certain preferred embodiments of the invention in detail, andis thus only representative and does not depict the actual scope of theinvention. Before describing the present invention in detail, it isunderstood that the invention is not limited to the particular aspectsand embodiments described, as these may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the invention defined by the appended claims.

Numerous methods are known for analyzing and sorting populations ofcells and other biomolecules, including methods based on cell size,density, or granularity in which separation is achieved bysedimentation, alone or in combination with density gradients andcentrifugation or elution. Other methods include those based ondifferential resistance of cells to osmotic lysis, as can be used, forexample, to separate white blood cells from whole blood. Furthermore,methods of depleting (i.e., reducing the number of) unwanted cells (orother biomolecules) from a more complex biological sample using specificantibodies that react with a cell surface marker can be used to removeor reduce the numbers of cells expressing that marker. Still other cellseparation methods include flow cytometry and magnetic cell sorting(e.g., using magnetic particle-conjugated antibodies), as well as othermethods that employ antibody affinity (or other high affinity bindingpairs) to particular biomolecules, including cell surface proteins.Using these technologies, positive enrichment or depletion ofparticularly desired, i.e., “targeted” or “target”, cell populations(i.e., those expressing a marker that can be targeted by the highaffinity binding moiety (e.g., an antibody, Fab fragment, receptor,etc.) conjugated to the labeled detection/separation reagent can beachieved.

Thus, this invention addresses the separation of one or more desired ortarget biomolecule species, particularly one or more target cellpopulations, from a more complex biological sample such as a cellularmixture (e.g., whole blood, a homogenized biopsy or tissue sample,etc.). A “target biological material” or “target biomolecule” refers toany biological substrate, for example, cells, organelles, and otherbiological materials, a user desires to isolate, enrich for, deplete, ortarget and for which a specific binding moiety (partner) can be preparedso as to specifically label or bind the material. The list of suitabletarget biomolecules is extensive, and includes microorganisms such asprotozoa, bacteria, yeast, and other fungi, cultured cells frommulti-celled organisms (including mammalian and other vertebrate cells,viruses, and fragments of the cells and viruses), eukaryotic cellpopulations that express one or more targetable cell surface antigens,and organelles or other subcellular structures (e.g., exosomes,proteasomes, ribosomes, etc.) that include a targetable protein or otherbiomolecule (e.g., a carbohydrate, lipid, etc.). Indeed, any biologicalmaterial (i.e., biomolecule), either a single molecule (e.g., a protein)or an organized or amorphous aggregate of one or more molecules (of thesame of different molecular species), that can be targeted by atargeting moiety can be isolated or purified using the nanomagneticparticles and methods of the invention.

The instant methods are based on the use of the new patentable class ofmagnetibuoyant separation techniques, which can be used to separatetargeted biomolecules (up to and including intact, viable cells) fromother components in a reaction mixture by magnetic cell separationtechniques. If desired, other separations can also be performed in orderto enrich or deplete one or more other biomolecule species (e.g., cellpopulations) present in the reaction mixture (as a result of beingpresent in the original sample to be analyzed). Indeed, in preferredmagnetibuoyant separations according to the invention, separation basedon the use of targeted buoyant microparticles (e.g., microbubbles andthe like) is used in conjunction with magnetic separation for parallelor serial processing of a biological sample in order to enrich for twoor more desired cell populations (or other biomolecule species) or toenrich for at least one target cell population (or other biomoleculespecies) and deplete another.

To perform magnetibuoyant separations according to the invention, bothmagnetic separation and buoyant separation processes are employed,either concurrently (in parallel) or one after another (in series; insome of these embodiments, magnetic separation may be performed beforebuoyant separation, while in others, the buoyant separation procedure isperformed before the magnetic separation procedure). The inventionenvisions the combined use of any suitable magnetic separation andbuoyant separation processes that are compatible in the particularapplication (with adaptations as appropriate). In preferred embodiments,the magnetic separation process utilizes targeted magnetic particles,particularly targeted nanomagnetic particles, and the buoyant separationprocess employs targeted buoyant particles, preferably targeted buoyantmicroparticles, e.g., buoyant microbubbles. Preferably, the targetingmoieties of the targeted magnetic particles target different biomoleculespecies than those biomolecules targeted by the targeting moieties ofthe targeted buoyant particles.

In practice, the magnetic separation utilizes a magnetic field gradientgenerated by a magnetic source, e.g., by a permanent magnet or anelectromagnet. Any type and form of magnet can be used. By applying amagnetic gradient to a reaction mixture containing an aliquot of abiological or cell sample, essentially all cells (i.e., 50-99% or more)bound by targeted magnetic particles (via an included targeting moietythat targets a biomolecule of interest, e.g., a protein expressed on thesurface of the targeted cell type(s)) can be separated from the othercomponents of the reaction mixture, whereas essentially all cells (i.e.,50-99% or more) bound by the targeted buoyant particles remain in thereaction mixture (unless such cells have already been removed from thereaction mixture using a buoyant separation process adapted for theparticular targeted buoyant particle species being used).

Targeting is provided by coupling magnetic particles or buoyantparticles to one or more targeting moieties. For a given targetedmagnetic particle species or targeted buoyant particle species of theinvention, one or more targeting moiety molecules of the particulartargeting moiety species can be coupled (directly or indirectly) to amagnetic particle or buoyant particle, as the case may be. Suchtargeting moiety molecule provides the capacity to specifically bind oneor more desired molecules of biomolecular species being targeted. Insome embodiments, a targeting moiety molecule has the capacity to bindtwo or more different desired (i.e., target) biomolecule species.Examples of such plural- or multi-specific targeting moieties includeantibodies, dendrimers, and the like engineered to target differentantigens (or different epitopes on the same antigen). In otherembodiments, a given targeted buoyant or magnetic particle species hastwo or more (e.g., 2-20) different targeting moiety species, preferablyeach targeting a different antigen (or different epitopes on the sameantigen), coupled thereto. Representative examples of biomolecules thatcan be targeted by the targeted buoyant and/or magnetic particle speciesof the invention include cell surface markers such as CD1c, CD2, CD3,CD4, CD7, CD8, CD11b, CD14, CD15, CD16, CD19, CD23, CD25, CD27, CD34,CD36, CD38, CD43, CD45, CD45RO, CD45RA, CD56, CD61, CD123, CD127, CD133,CD193, CD235a, CD335, CD304, anti-Fc-epsilon, anti-T cell receptoralpha/beta, anti-T cell receptor gamma/delta, anti-Biotin, anti-IgE,anti-HLA-DR, and combinations thereof. Particularly preferred proteinsthat can be targeted, for example, by a monoclonal antibody specificallyreactive therewith, to separate target cell populations from biologicalsamples include the following cell surface proteins:

Human Mouse Specificity Specificity CD4 CD4 CD8 CD8 CD19 CD19 CD14 CD11cCD56 CD25 CD25 Ter119 CD235 CX3CR1 Epcam/CD326 CD20 TSPAN33 CD20 Lfr5ERBB2/HER2 GPR35/CXCR8

In the context of the invention, targeted separation (for enrichment ordepletion) is achieved through the use of a targeting moiety conjugatedto the separable particle (e.g., a nanomagnetic particle of theinvention, a conventional magnetic particle, a buoyant particle (e.g., amicrobubble), etc.). The targeting moiety is typically a high affinitybinding reagent that can be conjugated to the separable particle by asuitable chemistry (preferably one involving covalent bonding that doesnot disrupt binding between the high affinity binding reagent and thetargeted biomolecule, preferably a protein expressed on the surface of atargeted cell population, organelle, or other biomolecule). Examples ofsuch high affinity binding reagents include members of high affinitybinding pairs. Such members include antibodies (particularly monoclonalantibodies), antigen-binding antibody fragments (e.g., Fab fragments),or another member of a high affinity bending pair (one of which isconjugated to the separable particle and the other of which is the“target” present on the biomolecule or structure being targeted). Insome embodiments, the high affinity binding reagent and/or separableparticle to which it is conjugated is labeled with a detectable agentsuitable for cell separation (e.g., FACS), such as a fluorescent dye.

High affinity binding reagents conjugated to separable (e.g., bymagnetic or electric fields, buoyancy, etc.) particles can be used toseparate desired biomolecules (e.g., a cell population expressing aparticular cell surface antigen) from other reaction mixture componentsunder conditions that allow the binding reagents to specifically bindtheir corresponding targets (e.g., antigens in the case of antibodies,antigen-binding antibody fragments, etc.).

The practice of the separation methods of the invention comprise thefollowing steps: in a reaction mixture, immobilizing the targetbiomolecule, for example, a target cell population expressing aparticular cell surface marker, present in a biological sample known orsuspected to contain the target biomolecule, which biomolecule isspecifically bound by the targeting moiety of a nanomagnetic particle ofthe invention in a ferromagnetic matrix through the use of a magneticfield; washing the matrix to remove unbound components in the reactionmixture; and removing the magnetic field to elute the targetedbiomolecule from the matrix. As a result, a target biomolecule (e.g., atarget cell population) is enriched; in addition or alternatively, thebiological sample is depleted of the target biomolecule (provided thatat the material washed from the matrix is retained for further use).Elution of material from the ferromagnetic matrix can be performed usinggravity flow, centrifugation, vacuum filtration, or a pressure gradient.

The term “magnetic separation” refers to separation procedures forconstituent components of complex samples, e.g., biological samples.Such procedures include magnetic separation mediated by targetingmoieties that comprise one member of a high affinity binding pair (e.g.,a monoclonal antibody that specifically binds a target cell surfaceantigen) conjugated or otherwise linked to a nanomagnetic particleaccording to the invention. Magnetic separation can be combined withother separation procedures, including those that employ targetedbuoyant particles and/or separation techniques known in the art thatalso rely on high affinity binding pairs (e.g., antibodies and theircognate antigens), for instance, affinity chromatography, “panning”(where one member of the high affinity binding pair is attached to asolid matrix (e.g., the well of a microtiter plate). Fluorescenceactivated cell sorting (FACS) can also be used if fluorescent tags areincluded in the targeted separable particles. Indeed, any now known orlater developed ligand-dependent separation technique can be used inconjunction with positive and/or negative separation techniques thatrely on physical properties of the target biomolecule rather thanaffinity, including filtration, size exclusion chromatography, anddensity gradient centrifugation.

The invention also includes kits for performing the magnetic separationmethods described herein, alone or in addition to other separationmethods. Such kits include targeted nanomagnetic particles of theinvention that target a desired biomolecule, for example, a cell surfaceantigen expressed on the surface of a particular cell type. The targetednanomagnetic particles are typically packaged in containers that includesuch quantities of the particles as are needed to perform one or moremagnetic separation procedures. Instructions (or a link or websiteaddress containing such instructions) for use of the targetednanomagnetic particles (and any other included reagent(s), e.g.,targeted buoyant microbubbles) are also typically included in any suchkit.

Magnetic Separation

Among techniques known for separating components of a biologicalmaterial or sample are those that make use of magnetic separationtechniques. Magnetic separation methods typically selectively retainmagnetic materials in a chamber or column disposed in a magnetic field.Such methods typically include passing a biological material or samplethrough one or more separation columns. Briefly, the biological materialor sample is magnetically labeled by attachment to targeted nanomagneticparticles of the invention through the use of a targeting moietyconjugated to the particles, which targeting moiety targets a desired(or “target”) biomolecule known or suspected to be present in thesample, for example, displayed on the surface of certain cells known orsuspected to be present in the same. A suspension of the labeled targetsample is then applied to the separation chamber or column. To separatethe targeted biomolecule species from the remainder of the reactionmixture, the targeted biological material is retained in the chamber inthe presence of a magnetic field. The retained targeted biologicalmaterial can then be eluted by changing the strength of, or byeliminating, the magnetic field.

In some embodiments, high gradient magnetic separation (HGMS) is used(Miltenyi et al., Cytometry, 11, 231 (1990)). In HGMS, a matrix ofmaterial of suitable magnetic susceptibility such as iron wool or steelbeads is placed in a chamber or column such that when a magnetic fieldis applied, a high magnetic field gradient is locally induced close tothe surface of the matrix, permitting retention of complexes of themagnetized particles and targeted biological material formed through theassociation of the members of the high affinity binding pairs present inthe mixture.

The targeted magnetic particles and methods of the invention can be usedfor the magnetic separation of, or to magnetically label and, ifdesired, isolate, any desired target substance or analyte (e.g., cells,organelles, etc.). Of particular interest is separating one or morespecific biomolecule(s) from a complex biological mixture. The presentinvention has great utility, in that almost any target substance may beseparated once a specific binding member for that substance isavailable. The targeting moiety can be any member of a specific,high-affinity binding pair, or a substance associated with a member of aspecific, high-affinity binding pair. For example, a cell surfaceantigen-antibody binding pair can be used to isolate the antigen itself,cells that express the antigen, a particular organelle involved inprocessing of the antigen, etc. The devices and methods of the presentinvention are also advantageously applied to diagnostic techniquesinvolving the binding of a receptor and ligand, such as immunoassays,and the like.

Targeted Magnetic Particles

Two classes of magnetic oxides, ferrites and non-ferrites, can be usedfor the production of the targeted magnetic particles of the invention,particularly targeted nanomagnetic particles (see commonly-owned,co-pending U.S. Ser. No. 15/143,552, filed 30 Apr. 2016, U.S. patentapplication pub. no. 20160320376, and WO 2016/179053). Ferrites, oriron-containing transition metal oxides, can generally be represented asXO.Fe₂O₃, where “X” may be Fe, Ni, Cr, Co, Mn, Mg, Mo, Gd, Cu, V, Dy,Ey, Tm, or Yb. Therefore, in the process of synthesizing magnetitesuperclusters, one would substitute the Fe²⁺-containing iron salt withone of the aforementioned divalent metal ion salts. The most preferablein this class of ferrites is FeO.Fe₂O₃, which is better known asmagnetite or Fe₃O₄. The non-ferrite class of magnetic oxides are void ofthe iron atom but instead are substituted with a combination of two ormore ions of these transition metals: Cr; Co; Mn; Ni; Mo; Gd; Dy; Ey;Tm; and Yb. Such non-ferrite-based magnetic oxides typically produce aspectrum of colored nanomagnetic particles but are less magneticallyresponsive than the ferrite class of magnetic oxides.

Magnetite crystals were first synthesized almost a century ago. Thesubsequent processing and stabilization of the magnetite crystals hasspawned many different types of magnetic particles of different sizes,with different surface coatings, and for different applications. Inpreferred embodiments, magnetite (Fe₃O₄) crystals are first synthesizedusing any suitable process, including the well-known aqueous basedco-precipitation method [Massart 1982, Schwertmann 1991]. Stoichiometricmixtures of ferrous (Fe²⁺) and ferric (Fe³⁺) iron salts are titratedwith a strong base under an inert atmosphere to yield 1 um-3 μm diametermagnetite crystals. Variables such as the mole-ratio of the iron salts(e.g., 1.0 M Fe²⁺: 2.0 M Fe³⁺ to 2.0 M Fe²⁺: 1.0 MFe³⁺), reactiontemperature (e.g., 40° C. to 95° C.), type of base counterions (e.g.,ammonium, sodium, potassium) used, and the rate of base addition (e.g.,2 mL/minute to 200 mL/minute) are optimized in order to produce thehighest quality ‘bare’ magnetite crystals. These magnetite crystals arenext sonicated at high power in order to yield quasi-stable 90 nm-110 nm(nanometer) sized nanomagnetic particles that are immediately silanizedusing an aqueous acidic silanization procedure concomitant with hightemperature dehydration in order to obtain silanized nanomagneticparticles.

Silanization can be accomplished using any suitable process. Forexample, after 25 minutes of sonication at high power (750 W) using a0.5 inch titanium probe tip, nanomagnetic particles are transferred intoa 3-neck round-bottom glass reaction vessel kept under nitrogen gascontaining 50 v % glycerol together with an overhead stirrer. A 10 wt %(relative to the iron mass) solution of sodium silicate is then added at0.5 ml/minute, followed immediately by the addition of 0.5 M glacialacetic acid at 1 mL/minute until a pH of 6 is attained. The temperatureis then raised to 180° C. and the mixture is allowed to dehydrate for atleast 2 hours, then cooled and washed using water.

In various preferred embodiments, the ‘bare’ magnetite crystals arefirst peptized using a strong metal ion chelating agent such as EGTA inorder to make available additional seed hydroxyl groups for condensationwith the silanization reagent. Peptization is achieved by sonicating the2 um size magnetite superclusters in the presence of the chelating agent(e.g., EGTA) in order to introduce additional hydroxyl groups onto themagnetite particles and afford greater colloidal stability. In yetanother preferred embodiment, two silanization reagents are usedsequentially in order to both enhance encapsulation as well as toprovide additional couplable groups by virtue of the inherentfunctionalities present in the secondary silanization reagent.Sequential silanization can be achieved, for example, by firstsilanizing sonicated magnetite particles using sodium silicate asdescribed above, followed immediately by the addition of an amino-silanesuch as aminopropyl-trimethoxysilane (APTS) prior to dehydration at 180°C. (see Example 2, below).

A second round of high power sonication, albeit brief, is performed inorder to reduce the particle size, preferentially to 95 nm-105 nm. Next,these silanized nanomagnetic particles are mixed with a heated solutioncontaining a protein/polymer mixture, for example, BSA (bovine serumalbumin) and the polysaccharide dextran (99 wt % BSA: 1 wt % dextran to50 wt % BSA: 50 wt % Dextran). This can be accomplished, for example, byheating a solution containing a mixture of BSA and Dextran to 70° C.just prior to mixing it with sonicated magnetite particles in a sealed3-neck reaction vessel under a nitrogen atmosphere. The coating processis allowed to proceed for 30 minutes. The suspension then is cooled andwashed using, for example, a high-field magnetic dipole separator.

Heating concentrated BSA solutions to temperatures in excess of 58° C.is known to produce irreversible aggregates of BSA mediated by theformation of disulphide bonds and hydrogen bonding of beta sheetsbetween individual BSA molecules [Wetzel, 1980]. In one preferredembodiment, maleimide groups are introduced into the BSA protein priorto mixing with the sonicated silanized particles in order to furtherpromote the formation of disulphide bonds. The BSA/Dextran coatednanomagnetic particles are then washed with the aid of strongdipole/quadrupole-type magnetic separators to remove excess coatingmaterials as well as to narrow the size distribution of the particles toa final size of about 110 nm. The initial wash supernatant from thismagnetic fractionation step contains a significant amount (˜50% by ironmass) of 30 nm-80 nm size BSA/Dextran coated nanomagnetic particles.Such smaller sized nanomagnetic particles can also be effectivelyutilized for magnetically capturing/purifying intracellular and/orextracellular targets such as, but not limited to, endosomes andexosomes, respectively. The BSA/Dextran coated nanomagnetic particles soproduced typically have a PDI of 0.1. This PDI number is a measure ofthe width of the particle size distribution and is obtainedautomatically during DLS based size measurements. Generally,polydispersity indices less than 0.1 are typically referred to as“monodisperse” particle suspensions. More precisely, PDI=the square ofthe standard deviation divided by the mean diameter and is adimensionless number. Bioaffinity ligands, i.e., “targeting moieties”,such as antibodies and/or streptavidin, are then conjugated to the 110nm diameter BSA/Dextran coated nanomagnetic particles using standardhetero/homo-bifunctional coupling chemistries. Streptavidin-coatednanomagnetic particles so prepared are further heat-treated with a highionic strength salt solution (1 M to 5 M NaCl) in order to stabilize thesurface coatings on the particles.

In some embodiments, the targeting moieties associated with a targetednanomagnetic particle of the invention are labeled with a detectablelabel, for example, a radioisotope or fluorescent molecule, in order torender the particles, or the particle/targeted cell (or otherbiomolecular structure) complexes detectable through the use of acomplementary label detection instrument or system. Such labels can beincluded in the magnetic core particle and/or in one or more of theouter layers of a nanomagnetic particle of the invention. In otherembodiments where particle/cell detection is desired, a technology fordetecting the particle's magnetic signal may be employed, arepresentative example of which is SQUID technology, which can be usedto detect magnetic labels by virtue of the magnetic fields that theyproduce [Clarke and Braginski, SQUID Handbook, vol. 1, (2004)].

Buoyancy-Based Separation

The present invention utilizes targeted magnetic particles and targetedbuoyant particles in combination for affinity isolation or separation ofdesired biomolecules, especially particular cell types, from biologicalsamples, cell cultures, and the like. Similar to targeted magneticparticles, targeted buoyant particles (e.g., microbubbles) employ atargeting moiety to target a desired biomolecule, e.g., an antigenpresent on the surface of a target cell type. The targeting moiety istypically a high affinity binding reagent that can be conjugated to thebuoyant particles by a suitable chemistry (preferably one involvingcovalent bonding that does not disrupt binding between the high affinitybinding reagent and the targeted biomolecule, preferably a proteinexpressed on the surface of a targeted cell population, organelle, orother biomolecule).

The buoyant microparticles, e.g., microbubbles, of the present inventionhave an advantage over solid particles for biomolecular separations inthat the normal force of gravity and the buoyant force of the buoyantmicroparticle are in different directions, thus resulting in asignificant reduction in non-specific binding and entrapment of speciesthat typically sink toward the bottom of a reaction vessel duringseparation. The separation can be enhanced with the unbound cells, forexample, being forced away from the microbubbles in a low centrifugalfield, as with a modest centrifugal speed, under conditions that do notadversely affect the microbubbles (or other buoyant microparticles).

As those in the art will appreciate, any suitable buoyant microparticlecan be adapted for use in practicing the invention. Exemplary buoyantmicroparticle species include protein microbubbles, such as albuminmicrobubbles. In some embodiments of the invention, the microbubbles areprotein microbubbles, which can be comprised of any peptide,polypeptide, protein, or combinations thereof. In some embodiments, theprotein is albumin. Both synthetic and naturally occurring peptides,polypeptides, proteins, and combinations are contemplated by theinvention. In some embodiments, protein microbubbles can be readilyformed into microbubbles through the introduction of a gas, for example,by introducing a gas into a protein-containing solution by sonication,heating, or other suitable process.

In other embodiments, the microbubbles are glass microbubbles,preferably glass microbubbles having a density of about 0.6 g/cc and anaverage diameter of about 30 microns. In some embodiments that use glassmicrobubbles, the glass microbubbles are treated to generate reactivesurface residues, which are in turn reacted with 3-aminopropyltriethoxysilane to generate amine functional groups. In still other embodiments,glass microbubbles can be cis-diol coated and the targeting moiety canbe directly coupled to the glass through the cis-diol coating. Thecis-diol coating can be generated, for example, by treating glassmicrobubbles to generate reactive surface hydroxyl residues, reactingthose residues with 3-glycidoxypropyltrimethoxysilane to generate epoxyfunctional residues, and then treating the epoxy functional residueswith acid to convert the epoxy function to cis-diol functions.

Herein, “bubble” refers to a small, hollow and lightweight globule,typically a small spherical volume of gas encased within a thin film.Bubbles can be filled with any gas, including, but not limited tooxygen, nitrogen, carbon dioxide, helium, fluorocarbon gases and variouscombinations thereof, such as air. The thin film can be any materialthat can encase a small volume of gas, such as an insoluble protein orlipid; a polymeric or non polymeric material; a solid such as a metal; asolid glass, ceramic or similar material; or a plastic, such aspolystyrene, polyethylene, polypropylene, nylon, etc. In some preferredembodiments, the thin film is albumin. In other preferred embodiments,the thin film is borosilicate glass. In some embodiments, the thin filmis stable under the conditions and solutions it is exposed to. In otherembodiments, the bubble can be selectively burst, crushed, orsolubilized. “Microbubbles” are small bubbles, generally in the range of0.1 to100 microns, typically 1 to 50, and frequently 2 to 20 or 2 to 30microns in diameter.

In use, the invention involves the steps of providing targeted magneticnanoparticles and targeted microbubbles in solution, contacting thetargeted magnetic nanoparticles and targeted microbubbles with abiological sample known or suspected to contain the biomolecule speciesof interest in solution under conditions that allow the targetingmoieties and targeted biomolecules to interact, thereby generatingbiomolecule:targeted magnetic particle and/or targeted microbubblecomplexes, and allowing the biomolecule:targeted microbubble complexesto float to the top of the solution, thereby separating the targetedbiomolecules complexed with the targeted microbubble from the othercomponents in the solution. The biomolecule:targeted microbubblecomplexes can then be isolated. After separation, thebiomolecule:targeted microbubble complexes can be treated with anysuitable process, e.g., detergent, pressure, or vacuum, to release thetargeted biomolecule species from the microbubble:biomolecule complexes.The biomolecule:targeted magnetic particle complexes can be isolatedwith a magnetic field device either separately or at the same time asthe biomolecule:microbubble complexes are separated.

Targeted Moieties for Magnetic Particles and Buoyant Microparticles

According to the invention, a targeting moiety is one member of a highaffinity binding pair, examples of which include receptors, receptorligands, aptamers, tetramers, and antibodies and antigen-bindingantibody fragments. Other suitable targeting moieties include biotin,avidin, and streptavidin.

In other embodiments, the targeting moiety can be indirectly coupled tothe magnetic nanoparticle or buoyant microparticle (e.g., microbubble),such as through the interaction of at least one other molecule. As anexample of indirect coupling, a magnetic nanoparticle or microbubble canbe directly coupled to streptavidin and the targeting moiety isbiotinylated. The particular targeted magnetic nanoparticle ormicrobubble species is formed by allowing the streptavidin and biotininteract to couple the targeting moiety to the magnetic nanoparticle ormicrobubble, thus forming a targeted magnetic nanoparticle or targetedmicrobubble species, as the case may be.

The targeting moiety can be directly coupled to the magneticnanoparticle or buoyant microparticle, for example, by using aheterobifunctional reagent such as sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, epoxy coating, or aminefunctional group on the magnetic nanoparticle or buoyant microparticle.

The term “targeting moiety” as used herein refers to any molecule thatis capable of specifically binding another molecule. In someembodiments, the targeting moiety is an antibody. In other embodiments,the targeting moiety is an antigen. In other embodiments, targetingmoieties can include, without limitation: nucleic acids (DNA, RNA, PNA,and nucleic acids that are mixtures thereof or that include nucleotidederivatives or analogs); cell-surface receptor molecules; ligands forcell-surface receptors; and biological, chemical, or other moleculesthat have affinity for another molecule, such as biotin and avidin. Thetargeting moieties of the present invention need not comprise an entirenaturally occurring molecule but may consist of only a portion,fragment, or subunit of a naturally or non-naturally occurring molecule,as. for example, the Fab fragment of an antibody. The targeting moietymay further comprise a marker or label that can be detected.

Targeting moieties can be generated by any suitable method. For example,antibodies can be found in an antiserum, prepared from a hybridomatissue culture supernatant or ascites fluid, or may be derived from arecombinant expression system, as is well known in the art. Fragments,portions, or subunits of, for example, an antibody, receptor or othermolecule, can be generated by chemical, enzymatic, or other techniques,yielding, for example, well-known (e.g., Fab, Fab′) or novel molecules.Antibodies can be monoclonal or polyclonal. The present invention alsocontemplates that targeting moieties can include recombinant, chimeric,and hybrid molecules, such as humanized and primatized antibodies, andother non-naturally occurring antibody forms. Those skilled in the artwill recognized that the non-limiting examples given above describingvarious forms of antibodies can also be extended to other targetingmoieties such that recombinant, chimeric, hybrid, truncated, etc., formsof non-antibody targeting molecules can be used in the methods of thepresent invention.

The terms “specifically binding”, “specific binding”, and the like meanthat an antibody or other molecule, especially a targeting moiety of theinvention, binds to a target such as an antigen, ligand or otheranalyte, with greater affinity than it binds to other molecules underthe specified conditions of the present invention. Additionally specificbinding can be directed by relatively high avidity molecules such astetramers, Pentamers and Dextramers that are used to increasespecificity and avidity of binding events. Antibodies or antibodyfragments, as known in the art, are polypeptide molecules that containregions that can bind other molecules, such as antigens. In variousembodiments of the invention, “specifically binding” may mean that anantibody or other targeting moiety binds to a target analyte molecule (abiomolecule) with at least about a 10⁶-fold greater affinity, preferablyat least about a 10⁷-fold greater affinity, more preferably at leastabout a 10⁸-fold greater affinity, and most preferably at least about a10⁹-fold greater affinity than it binds molecules unrelated to thetarget molecule. Typically, specific binding refers to affinities in therange of about 10⁶-fold to about 10⁹-fold greater than non-specificbinding. In some embodiments, specific binding may be characterized byaffinities greater than 10⁹-fold over non-specific binding. Whenever arange appears herein, as in “1-10 or one to ten, the range referswithout limitation to each integer or unit of measure in the givenrange. Thus, by 1-10 it is meant each of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and any subunit in between.

Antibody fragments, e.g., Fab and F(ab′)₂ fragments, single chainantibodies, etc. that recognize specific epitopes may be generated byknown techniques.

“Antigens” are macromolecules capable of generating an antibody responsein an animal and being recognized by the resulting antibody. Antigenstypically comprise at least one antigenic determinant or “epitope,”which is the region of the antigen that is bound by the antibody.

A “receptor” or “biological receptor” typically refers to a molecularstructure within or on the surface of a cell characterized by selectivebinding of a specific substance (e.g., a “ligand”) that results in aspecific physiologic effect that accompanies the binding. Examples ofreceptors include cell-surface receptors for biomolecules such as, forexample, peptide hormones, neurotransmitters, antigens, complementfragments, and immunoglobulins, and cytoplasmic receptors for steroidhormones. As used herein, however, the receptor will typically beisolated and purified and need not effect or be capable of effecting aphysiological or other biological effect, other than targeting the othermember of the high affinity binding pair to which it belongs.

The term “ligand” refers generally to a molecule that binds to areceptor. Typically, a ligand is a small, soluble biomolecule, such as apeptide, hormone or neurotransmitter.

As will be appreciated, coating of magnetic nanoparticles and buoyantmicroparticles with a targeting moiety can be accomplished by any methodknown in the art. Advantageously, proteins contain amine functionalgroups that can serve as the basis for numerous modifications andcoupling reactions, such as reactions with aldehydes. Furthermore, thematerials that make up the magnetic nanoparticles and buoyantmicroparticles useful in practicing the invention, e.g., protein, glass,phospholipid and the like, can be chemically derivatized orfunctionalized to covalently interact with various types of targetingmoieties. Indeed, a variety of commercial reagents, products, and kitsfor coupling proteins and other targeting moieties molecules to themagnetic nanoparticles and buoyant microparticles of the invention areknown in the art.

In other embodiments of the invention, a targeting moiety can beindirectly coupled to the magnetic nanoparticles and buoyantmicroparticles, such as through the interaction of at least one othermolecule. For example, magnetic nanoparticles and/or buoyantmicroparticles can be directly coupled to streptavidin and the targetingmoiety is biotinylated, such that the streptavidin and biotin interactto couple the targeting moiety to the particles. See e.g., Avidin-BiotinChemistry: A Handbook (Savage, et. al., eds. Pierce Chemical Co.,Rockford, Ill., 1992).

In Vivo Applications

The magnetibuoyant separation methods of the invention can be adaptedfor many in vivo diagnostic and therapeutic uses, including imaging,cell therapies, and delivery of therapeutic agents to cells. Combiningthe use of targeted magnetic particles with targeted buoyancy-basedtechniques provides even greater advantages such as increasing thepurity of the isolated biomolecules, reducing sample processing times,and yielding one or more specific enriched cell populations from asingle complex sample. As will be appreciated, in some embodiments, acell, while not present in a tissue, is present in a population ofcells. As used herein, a “population of cells” or “cell population”refers to a group of at least two cells, e.g., 2-10 cells, 2-100 cells,2-1000 cells, 2-10,000 cells, 2-100,000 cells, 2-10⁶, 2-10⁷, 2-10⁸, orany value in between, or more cells. Optionally, a population of cellscan be cells that have a common origin, e.g., descended from the sameparental cell, isolated from or descended from cells isolated from thesame tissue, or isolated from or descended from cells isolated from thesame tissue sample. A population of cells can comprise one or more celltypes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell types or more celltypes. A population of cells can also be heterogeneous or homogeneous. Acell population can be substantially homogeneous if it comprises atleast about 90% of the same cell type, e.g., 90%, 92%, 95%, 98%, 99% ormore of the cells in the population are of the same cell type. Apopulation of cells can be heterogeneous if less than about 90% of thecells present in the population are of the same cell type.

Cells, or cell populations, separated is accordance with the methods ofthe invention can be administered to patients or subjects via anysuitable route in order to treat a variety of conditions, diseases, anddisorders. Preferably, the subject treated with cells isolated per theinvention is a mammal. The mammal can be a human, non-human primate,mouse, rat, dog, cat, horse, or cow, but is not limited to theseexamples. Mammals other than humans can be advantageously used assubjects that represent animal models of a disease associated with adeficiency, malfunction, and/or failure of a given cell, tissue, ororgan or a deficiency, malfunction, or failure of a stem cellcompartment. In addition, the methods described herein can be used totreat domesticated animals and/or pets. A subject can be male or female.A subject can be one who has been previously diagnosed with oridentified as suffering from or having a deficiency, malfunction, and/orfailure of a cell type, tissue, organ, or stem cell compartment or oneor more diseases or conditions associated with such a condition, andoptionally, but need not have already undergone treatment for such acondition. A subject can also be one who has been diagnosed with oridentified as suffering from a condition including a deficiency,malfunction, and/or failure of a cell type or tissue or of a stem cellcompartment, but who shows improvements in known risk factors as aresult of receiving one or more treatments for such a condition.Alternatively, a subject can also be one who has not been previouslydiagnosed as having such a condition. For example, a subject can be onewho exhibits one or more risk factors for such a condition or a subjectwho does not exhibit risk factors for such conditions.

Cell Therapy

Today, many human diseases cannot be satisfactorily treated withstandard pharmaceuticals. For some of these diseases, cell therapiesoffer an attractive alternative. Cell-based therapies generally requiresignificant handling and processing of cellular products. Current celltherapy methods require substantial infrastructure and equipment to meetmanufacturing and regulatory requirements, including good manufacturingpractices, which involve the use of suitable clean rooms and personnelto maintain rooms, devices, production, quality control, and qualityassurance procedures under conditions that ensure non-contamination ofsamples to maintain sterility. Cell-based products are typicallyprocessed using a combination of different devices and disposables.Transfer of products and reagents in such processes can be manual and/orautomated.

Magnetibuoyant cell separation can include both enrichment and depletionprocedures. If target cells can be identified using cell surfaceproteins (or other cell surface biomolecules), they can be enriched tohigh purity through one or more rounds of enrichment and/or depletion.In other cases, target cells can be identified and removed from theresulting cellular product, which may be a heterogeneous mixture ofdifferent desired cells in which the number of cells targeted forremoval has been reduced. Of course, combinations of both enrichment anddepletion can be used.

Magnetibuoyant labeling of cells using targeted (nano)magnetic particlesand targeted buoyant particles (e.g., microbubbles) of the inventionincludes at least one suitable targeting moiety, typically a specificbinding member of a high affinity binding pair, for each of magneticparticle species and each buoyant particle species, wherein thetargeting moieties for the different particle types target differentbiomolecules (e.g., cell surface receptors). The target cell/particlecomplexes can then be isolated using a magnetic separation device,preferably also in conjunction with using targeted buoyant particles tofurther enrich the desired cell population(s). The isolation ofmultipotent cells, e.g., hematopoietic stem or progenitor cells, is ofparticular interest, although the present invention can be applied to awide range of cell types or other biological materials or samples.

Cellular products produced in accordance with the invention can be usedin therapy immediately or stored for later use using known methods.Formulation steps include adjusting the separated cell-containingpreparation to a desired volume or cell concentration, exchangingprocessing liquids with injectable solutions, adding stabilizers (e.g.,autologous plasma or serum, serum albumins, other proteins or syntheticpolymers, etc.) or adjuvants, supplementation with cryoprotective agentssuch as DMSO for subsequent storage, drawing of retention aliquots forquality control, delivery to combinations of bags or syringes forinfusion, etc. Exemplary cell therapies that can be practiced using cellpopulations enriched in accordance with the invention include stem celltransplantation for tissue regeneration, including to treat myocardialinfarction, liver damage, or neurodegenerative diseases; to treatrefractory autoimmune diseases such as systemic lupus erythematosus,systemic scleroderma, type 1 diabetes, or multiple sclerosis; to treatcancer, for example, leukemia (including acute myeloid leukemia, chronicmyeloid leukemia, acute lymphocytic leukemia, chronic lymphocyticleukemia); immunotherapy to treat cancer, viral and bacterialinfections, etc.

Preferred cell types that can be enriched per the invention include stemcells. Stem cellsare unspecialized cells that have the capacity toself-renew and to differentiate into specialized cell types, such asneurons, liver, or muscle cells. Embryonic stem cells (ESCs) and inducedpluripotent stem cells (iPSCs) are pluripotent stem cells (PSCs) thathave the potential to differentiate and become any cell type found inthe human (or other animal species) body. Adult stem cells such asneural stem cells (NSCs), mesenchymai stem cells (MSCs), andhematopoietic stem cells (HSCs) are multipotent stem cells, whosedifferentiation is limited to the cell types found in the tissue oforigin. A wide range of markers, particularly cell-surface markers, areknown for these types of stem cells, which markers (whether now known orlater discovered) can serve as target: for the targeting moieties usefulin practicing the invention. Similarly, markers known to be absent fromcells intended for a cell-based therapy can be used to deplete cellsexpressing such markers from cell populations prior to administration toa subject (e.g., a human) to be treated.

Importantly, the targeted magnetic and microbubble buoyant particles ofthis invention can be sterilized using any suitable method, includingfilter sterilized (due to the particles' small size) for use intherapeutic and/or in-vivo/in-vitro procedures where sterile processingis mandated or desired.

As described above, the cells separated by using the magnetibuoyantseparation methods and compositions of the invention can be any type ofcell, e.g., an adult cell, an embryonic cell, a differentiated cell, astem cell, a progenitor cell, and/or a somatic cell. The separated cellscan be obtained from a subject, e.g., from a biological sample obtainedfrom a patient, from cell culture, or any other suitable source. In someembodiments, the cells are mammalian cells, for example, human cells. Insome embodiments, the cells are adult cells. In other embodiments, thecells are neonatal cells, fetal cells, amniotic cells, or cord bloodcells. The cells may be autologous or allogeneic, and they may or maynot be genetically modified or otherwise engineered.

The term “somatic cell” refers to any cell other than a germ cell, acell present in or obtained from a pre-implantation embryo, or a cellresulting from proliferation of such a cell in vitro. Stated anotherway, a somatic cell refers to any cells forming the body of an organism,as opposed to germline cells. In mammals, germline cells (also known as“gametes”) are the spermatozoa and ova that fuse during fertilization toproduce a cell called a zygote, from which the entire mammalian embryodevelops. Every other cell type in the mammalian body—apart from thesperm and ova, the cells from which they are made (gametocytes), andundifferentiated stem cells—is a somatic cell; internal organs, skin,bones, blood, and connective tissue are all made up of somatic cells. Insome embodiments, the somatic cell is a “non-embryonic somatic cell,” bywhich is meant a somatic cell that is not present in or obtained from anembryo and does not result from proliferation of such a cell in vitro.In some embodiments, the somatic cell is an “adult somatic cell,” bywhich is meant a cell that is present in or obtained from an organismother than an embryo or a fetus or results from proliferation of such acell in vitro. In this context, “adult” refers to tissues and cellsderived from or within an animal subject at any time after birth, and“embryonic” refers to tissues and cells derived from or within an animalsubject at any time prior to birth. It is noted that adult and neonatalor embryonic cells can be distinguished by structural differences, e.g.,epigenetic organization such as methylation patterns. In someembodiments, the somatic cell is a mammalian somatic cell. In someembodiments, the somatic cell is a human somatic cell. In someembodiments, the somatic cell is an adult somatic cell. In someembodiments, the somatic cell is a neonatal somatic cell.

A “differentiated cell” refers to a cell that is more specialized in itsfate or function than at a previous point in its development, andincludes both cells that are terminally differentiated and cells that,although not terminally differentiated, are more specialized than at aprevious point in their development. The development of a cell from anuncommitted cell (for example, a stem cell), to a cell with anincreasing degree of commitment to a particular differentiated celltype, and finally to a terminally differentiated cell, is known asprogressive differentiation or progressive commitment. In the context ofcell ontogeny, the adjective “differentiated”, or “differentiating”, isa relative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. Thus, stem cells can differentiate to lineage-restrictedprecursor cells (such as a mesodermal stem cell), which in turn candifferentiate into other types of precursor cells further down thepathway (such as a cardiomyocyte precursor), and then to an end-stagedifferentiated cell, which plays a characteristic role in a certaintissue type, and may or may not retain the capacity to proliferatefurther.

The term “stem cell” refers to a cell in an undifferentiated orpartially differentiated state that has the property of self-renewal andhas the developmental potential to naturally differentiate into a moredifferentiated cell type, without a specific implied meaning regardingdevelopmental potential (i.e., totipotent, pluripotent, multipotent,etc.). By “self-renewal” is meant that a stem cell is capable ofproliferation and giving rise to more such stem cells, while maintainingits developmental potential. Accordingly, the term “stem cell” refers toany subset of cells that have the developmental potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retain the capacity, under certaincircumstances, to proliferate without substantially differentiating. Theterm “somatic stem cell” is used herein to refer to any stem cellderived from non-embryonic tissue, including fetal, juvenile, and adulttissue. Natural somatic stem cells have been isolated from a widevariety of adult tissues including blood, bone marrow, brain, olfactoryepithelium, skin, pancreas, skeletal muscle, and cardiac muscle.Exemplary naturally occurring somatic stem cells include, but are notlimited to, mesenchymal stem cells and hematopoietic stem cells. In someembodiments, the stem or progenitor cells can be embryonic stem cells.As used herein, “embryonic stem cells” refers to stem cells derived fromtissue formed after fertilization but before the end of gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10-12 weeksgestation. Most frequently, embryonic stem cells are totipotent cellsderived from an early embryo or blastocyst. Embryonic stem cells can beobtained directly from suitable tissue, including, but not limited tohuman tissue, or from established embryonic cell lines.

Exemplary stem cells include embryonic stem cells, adult stem cells,pluripotent stem cells, neural stem cells, liver stem cells, muscle stemcells, muscle precursor stem cells, endothelial progenitor cells, bonemarrow stem cells, chondrogenic stem cells, lymphoid stem cells,mesenchymal stem cells, hematopoietic stem cells, central nervous systemstem cells, peripheral nervous system stem cells, and the like.

As used herein, “progenitor cells” refers to cells in anundifferentiated or partially differentiated state and that have thedevelopmental potential to differentiate into at least one moredifferentiated phenotype, without a specific implied meaning regardingdevelopmental potential (i.e., totipotent, pluripotent, multipotent,etc.) and that does not have the property of self-renewal. Accordingly,the term “progenitor cell” refers to any subset of cells that have thedevelopmental potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype. In someembodiments, the stem or progenitor cells are pluripotent stem cells. Insome embodiments, the stem or progenitor cells are totipotent stemcells.

The term “totipotent” refers to a stem cell that can give rise to anytissue or cell type in the body. “Pluripotent” stem cells can give riseto any type of cell in the body except germ line cells. Stem cells thatcan give rise to a smaller or limited number of different cell types aregenerally termed “multipotent.” Thus, totipotent cells differentiateinto pluripotent cells that can give rise to most, but not all, of thetissues necessary for fetal development. Pluripotent cells undergofurther differentiation into multipotent cells that are committed togive rise to cells that have a particular function. For example,multipotent hematopoietic stem cells give rise to the red blood cells,white blood cells, and platelets in the blood.

The term “pluripotent” refers to a cell with the capacity, underdifferent conditions, to differentiate to cell types characteristic ofall three germ cell layers (i.e., endoderm (e.g., gut tissue), mesoderm(e.g., blood, muscle, and vessels), and ectoderm (e.g., skin and nervecells)). Pluripotent cells are characterized primarily by their abilityto differentiate to all three germ layers, using, for example, a nudemouse teratoma formation assay. Pluripotency is also evidenced by theexpression of embryonic stem (ES) cell markers, although the preferredtest for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and non-limiting examples of multipotent cells can includeadult stem cells, such as, for example, hematopoietic stem cells andneural stem cells. “Multipotent” means a stem cell may form many typesof cells in a given lineage, but not cells of other lineages. Forexample, a multipotent blood stem cell can form the many different typesof blood cells (red, white, platelets, etc.), but it cannot formneurons. The term “multipotency” refers to a cell with the degree ofdevelopmental versatility that is less than totipotent and pluripotent.

The term “totipotency” refers to a cell with the degree ofdifferentiation describing a capacity to make all of the cells in theadult body as well as the extra-embryonic tissues, including theplacenta. As is known, fertilized eggs (zygotes) are totipotent, as areearly cleaved cells (blastomeres).

The cells used in the methods described herein can be cells that are notpresent in a tissue. As used herein, a “tissue” refers to an organizedbiomaterial (e.g., a group, layer, or aggregation) of similarlyspecialized cells united in the performance of at least one particularfunction. When cells are removed from an organized superstructure, orotherwise separated from an organized superstructure that exists invivo, they are no longer present in a tissue. For example, when a bloodsample is separated into two or more non-identical fractions, or aspleen is minced and mechanically disassociated with Pasteur pipettes,the cells are understood to no longer be present in a tissue. In someembodiments, cells that are not present in a tissue are isolated cells.The term “isolated” as used herein in reference to cells refers to acell that is mechanically or physically separated from another group ofcells with which they are normally associated in vivo. Optionally, anisolated cell may have been cultured in vitro, e.g., in the presence ofother cells.

Cell therapies that can be practiced in accordance with the inventioninclude graft engineering, e.g., in conjunction with, for example, stemcell or organ transplantation; immunotherapy to bacterial or viralinfections or cancer, e.g., by administering T cells to treat solidtumors (e.g., renal cell carcinoma, breast cancer, melanoma, pancreaticcancer, ovarian cancer, colorectal cancer, etc.) via cell-mediatedimmunity as well treating leukemia (e.g., acute myeloid leukemia,chronic myeloid leukemia, acute lymphoid leukemia, chronic lymphoidleukemia, etc.); treatment of refractory autoimmune diseases such assystemic lupus erythematosus or systemic scleroderma, type 1 diabetes,multiple sclerosis; treatment of infectious diseases; tissueregeneration (e.g., to treat myocardial infarction, liver damage, orneurodegenerative diseases); and tolerance induction (e.g., for use inconjunction with tissue or organ transplantation, to treat autoimmunediseases, etc.). Among the immunotherapeutic approaches of the inventionis adoptive cell therapy (ACT), which uses T cells that have beengenetically modified (or engineered) to express chimeric antigenreceptors (CARs) on their cell surfaces to allow the T cells torecognize a specific protein cell-surface antigen on cells of a tumorafflicting the subject, or, in other embodiments, of cell-surfaceantigens specific to a bacterial or viral infection. Typically, afterbeing engineered to target a cell-surface antigen expressed on diseasedcells (e.g., by a particular cancer cell type or cells infected by aparticular bacteria or virus), the CAR-T cells are expanded in cultureuntil they number in billions (or more), after which they can beadministered to a subject.

Magnetibuoyant cell separation methods can comprise both enrichment anddepletion procedures. If target cells can be identified based on surfaceproteins, target cells can be enriched to high purity. In somesituations, non-target cells can be identified based on their unwantedfunctional characteristics within a specific clinical context. Thesenon-target cells can be removed from the cellular product, resulting ina heterogeneous mixture of different target cells. For example, cellproducts processed by the present invention for graft engineering can beenriched for CD34 and/or CD133 or depleted for CD3, CD3 and CD19, CD6,CD4 and CD8, T Cell Receptor alpha/beta (TCR alpha/beta) orCD3/CD19/CD16/CD14, resulting either in enriched stem cell preparationsor stem cells supplemented with other immune cells such as NaturalKiller (NK) cells and dendritic cells.

In other embodiments, cell products prepared in accordance with thepresent invention for cellular therapy can be enriched, for example, forCD14 (monocytes), CD56 (natural killer cells), CD335 (NKp46, naturalkiller cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD1c(BDCA-1, blood dendritic cell subset), CD303 (BDCA-2), CD304 (BDCA-4,blood dendritic cell subset), NKp80 (natural killer cells, gamma/delta Tcells, effector/memory T cells), “6B11” (Va24Nb11; invariant naturalkiller T cells), CD137 (activated T cells), CD25 (regulatory T cells),or depleted for CD138 (plasma cells), CD4, CD8, CD19, CD25, CD45RA,CD45RO. In other embodiments, cell populations such as Natural Killercells, T cells, and the like can be used as effector cells in donorlymphocyte infusion approaches to eliminate virus or bacteria infectedcells, tumor cells, etc. Dendritic cells, either generated frommonocytes in cell culture or directly isolated, can be used to, forexample, to “vaccinate” patients to promote antigen-specific and naturalimmunity against virus infected cells, tumor cells, bacteria, and/orfungi.

Advantageously, the present invention allows for manufacturing ofcellular products sorted by magnetic and buoyant separation techniquesfor two or more different biomolecule species that can be performed in asingle reaction, thus avoiding potential harm (e.g., infection,contamination, increased temperature) to the desired cell product.Representative two parameter sorting applications include generatinghighly enriched regulatory T cells (e.g., the cell product may first bedepleted for CD8 and/or CD19 and/or CD49d and enriched for CD25), highlyenriched natural killer cells (e.g., CD3 depleted, CD56 enriched), andhighly enriched blood dendritic cell subsets (e.g., CD19 depleted, CD1cenriched).

In Vitro Applications

The magnetibuoyant methods and compositions of the invention can also beadapted for many in vitro diagnostic uses, in addition to therapeuticuses (as described above and elsewhere herein). Magnetic particles havebeen used in the past to isolate or enrich eukaryotic cells, bacterialspecies, nucleic acids, and proteins. Beside particle isolation or cellseparation, magnetic particles have also been used to stimulate oractivate cells by coating cell-activating ligands on the particlesurface so that full three-dimensional aspects of target engagement,often important in biological systems, are more accurately reproduced ascompared to solution phase activation protocols. In recent years,magnetic particles have been studied for use in newer in vitro tests. Inthe context of magnetic nanoparticles, examples of such diagnostic usesinclude evaluation of the potential health effects of nanomagneticparticles (Kevin, et al., Biosensors and Bioelectronics, 43, 88 (2013))and of nanotechnology-based systems for delivery of si-RNA (Dim, et al,J. Nanobiotechnology, 13, 61 (2015)). Nanoparticles are also in researchand development testing for application as targeted heating componentsthat can develop localized magnetic hyperthermia conditions for thetreatment of cancer (Makridis, et al., Mater Sci Eng C Mater Biol Appl.,63, 663 (2016).

Kits

A further object of the invention to provide compositions for isolating,enriching, recovering, and/or separating therapeutically,diagnostically, or scientifically valuable cells from a biological orcell sample, for example, peripheral blood, umbilical cord blood, and/orbone marrow. Such compositions include at least one targeted magneticparticle species and at least one targeted buoyant particle species.Such compositions can be separate or combined. Such compositions can bein liquid (e.g., prepared as solutions or suspensions) or dry (e.g., asa lyophilized powder to be reconstituted in an suitable buffer prior touse) form. Typically such compositions are provided in kit form, witheach kit preferably containing the components necessary to perform amagnetibuoyant separation of the desired biomolecule, e.g., a particularcell species or type from a cell-containing sample in accordance withthe methods described herein.

A “kit” in accordance with the invention may, by way of example,comprise at least one container having disposed therein a targetednanomagnetic particle species that targets a first biomolecule speciesof interest and another container having disposed therein a targetedbuoyant microparticle species that targets a second biomolecule speciesof interest (the first and second biomolecule species being different).In some embodiments, the targeted nanomagnetic particle species andtargeted buoyant microparticle species are disposed in the samecontainer. In other embodiments, the kit comprises containers thatcontain one or more different targeted nanomagnetic particle speciesand/or targeted buoyant microparticle species. A kit according to theinvention may further comprise other containers comprising one or moreof the following: buffers, solutions, or other reagents and materialsnecessary for performing magnetibuoyant separations; and vessels forconducting magnetibuoyant separations (e.g., centrifuge tubes, columns,etc.). Preferably, the kits of the invention further compriseinstructions for performing magnetibuoyant separations. Such kits, ifintended for diagnostic or therapeutic use, may also includenotification of a FDA approved use and instructions therefor.

EXAMPLES

The following Examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in its practice.These Examples are in no way to be considered to limit the scope of theinvention in any manner, and those having ordinary or greater skill inthe applicable arts will readily appreciate that the specificationthoroughly describes the invention and can be readily applied to carryout the objects and obtain the ends and advantages mentioned, as well asthose inherent therein.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. Furthermore, unlessotherwise stated, the experiments described in the Examples wereperformed using standard procedures, as described, for example, inSambrook, et al., Molecular Cloning: A Laboratory Manual (3 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1995); Current Protocols in CellBiology (CPCB) (Juan S. Bonifacino, et. al. ed., John Wiley and Sons,Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R.Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal CellCulture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather andDavid Barnes editors, Academic Press, 1st edition, 1998) which are allincorporated by reference herein in their entireties.

1. Synthesis of Silanized BSA/Dextran-Coated Nanomagnetic Particles

This example describes a preferred method for synthesizing silanizedBSA/Dextran coated nanomagnetic particles for use in the invention. Thissynthesis is carried out in three stages and involves first, thesynthesis of the bare (uncoated) magnetite superclusters followed by thesilanization of these superclusters, and, finally, a protein/polymercoating step using a mixture of BSA/Dextran.

Briefly, 5.02 g of ferrous sulfate and 7.22 g of ferric sulfate(SIGMA-ALDRICH; St. Louis, Mo.) are separately dissolved in 25 mL ofdegassed deionized water and then added into a reaction vesselcontaining 250 mL of degassed deionized water at 70° C. with continuousstirring. Next, 35 mL of 10 M ammonium hydroxide (SIGMA-ALDRICH; St.Louis, Mo.) is added into the reaction flask at a rate of 9 mL/minute,and the formation of the magnetite superclusters is allowed to proceedfor 30 minutes. The precipitate is then exhaustively washed withdeionized water using an in-house built ceramic low-gradient magneticseparator (LGMS) and finally stored under a nitrogen cap in degasseddeionized water. These magnetite superclusters typically have ahydrodynamic diameter in the range of 2 to 3 um as measured by dynamiclight scattering (Malvern Nano-S ZetaSizer; Westborough, Mass.).

Next, 1.65 g of the magnetite superclusters are sonicated in a 100 mLvolume of low ionic strength phosphate buffer (ACS grade monosodiumphosphate having a molecular weight of 137.99 g/mole) with the aid ofthe VCX750 Ultrasonic processor (Sonics & Materials Inc., Newtown,Conn.) using a cooled, jacketed reaction beaker to a final size of ˜110nm and then immediately transferred into a reaction flask contained in asilicone oil-bath. Next, 2.5 mL of a 66 mg/mL of sodium silicate (SiO₂⁻) solution (SIGMA-ALDRICH; St. Louis, Mo.) is added into the reactionflask at a rate of 0.5 mL/minute followed by acidification with ˜8 mL of0.5M acetic acid added at 1 mL/minute to a final pH value of 6.0. Thetemperature of the oil-bath is then raised to 170° C. and the particlesuspension is allowed to dehydrate for about 3 hours in order to promotethe surface silanization of the nanomagnetic particles. After cooling toroom temperature, the particle suspension is placed into a LGMS magneticseparator for 30 minutes and the magnetically pelletized particles arerecovered and washed exhaustively with a low ionic strength HEPES buffer(VWR, Visalia, Calif.). These silanized or SiO₂-derivatized magnetiteclusters typically have a hydrodynamic size of about 200 nm and dissolvemuch more slowly in strong acid than their non-silanized versions (seeTable 1, below).

To prepare the BSA/Dextran coated nanomagnetic particles, 1.4 g of thesilanized magnetite clusters are sonicated in a 135 mL volume of lowionic strength phosphate buffer with the aid of the VCX750 Ultrasonicprocessor (Sonics & Materials Inc.; Newtown, Conn.) using a cooled,jacketed reaction beaker to a final size of ˜100 nm and then immediatelytransferred into a 1 L jacketed reaction flask thermostated to 70° C.that contains 400 mL of a mixture of 20 mg/mL BSA (Lampire Biologicals;Pipersville, Pa.) and 0.2 mg/mL Dextran (SIGMA-ALDRICH; St. Louis, Mo.).This coating reaction is allowed to proceed for 30 minutes at 70° C. Thecoated nanomagnetic particles are then cooled to room temperature andleft undisturbed overnight at 4° C.

Next, the particles are slowly decanted from the vessel with the aid ofa low-field ceramic magnet held at the bottom of the vessel in order tosediment away the large size (˜300 nm) particle aggregates. Thecollected supernatant (of ˜100 nm diameter) is then subjected to 7cycles of high-field magnetic washes in low-ionic strength HEPES buffer(VWR; Visalia, Calif.). These high-field washes, in addition to removingthe excess reactants, also serves to significantly narrow the particlesize distribution to values of PDI. The final hydrodynamic particle sizeis typically about 115 nm. The overall yield starting from 1.65 g of themagnetite superclusters is typically at least 50%. The first high-fieldmagnetic wash supernatant, which typically has a hydrodynamic size of˜70 nm and which constitutes ˜35% of the total particle yield, iscollected as a by-product and can be used to produce smaller size (<100nm) nanomagnetic particle products for use as an in-vivo/in-vitrotracking/capture label as well as for magnetic cell isolations inconcert with HGMS columns (see EXAMPLE 3, below).

Finally, a member of a bioaffinity ligand pair such as Streptavidin,antibodies, or other desirable ligands can be covalently conjugated tothe ample BSA-derived functional groups present on these BSA/Dextrancoated nanomagnetic particles using standard hetero/homo-bifunctionalconjugation chemistries as will be familiar to those skilled in the art.

2. Synthesis of Nanomagnetic Particles by Peptization and Other Types ofSilanizing Agents

Electrolytes such as the chelating agents known more popularly as EDTA,EGTA, as well as weak bases and acids are referred to as peptizingagents in instances where they help to disperse precipitates intocolloidal sols. In this example, EGTA (SIGMA-ALDRICH; St. Louis, Mo.),which is a strong iron chelating agent, is added (at 0.25 molesEGTA/mole iron) immediately after the formation of the magnetitesuperclusters as in Example 1, above. This chelation step is allowed toproceed for 1 hour at 70° C. prior to washing up the magnetitesuperclusters as in Example 1, above. Unlike the ˜2.5 um size of thestarting magnetic superclusters, these EGTA peptized magnetite clusterstypically have a hydrodynamic diameter of about 1 um, and such a sizereduction is indicative of a successful dispersion of the magnetitesuperclusters.

In another embodiment, a sequential silanization method is used wherebyEGTA peptized magnetite superclusters are sonicated and silanized as inExample 1, above, and immediately after the addition of the sodiumsilicate solution, an equimolar amount of the amino-functionalizedsilanizing agent aminopropyltrimethoxysilane or APS (SIGMA-ALDRICH; St.Louis, Mo.) is added and the particles allowed to dehydrate for 90minutes at 160° C. after acidification to pH 6.0. Silanization has alsobeen achieved using just APS in lieu of sodium silicate as in Example 1,above, and the dehydration step allowed to proceed for 75 minutes at160° C.

Another silanization agent, hydroxymethyltriethoxysilane (Gelest,Morrisville, Pa.), is very hydrophilic, and has also been successfullyused to produce silanized nanomagnetic particles useful in the contextof the invention. In this particular case, 15 wt % of this silanizingagent (relative to the iron content) was used and the dehydration wasallowed to proceed for 2 hours at 160° C.

All of the aforementioned silanized magnetic particles have beensuccessfully coated with BSA/Dextran mixtures as described in Example 1,above. These types of silanized nanomagnetic particles, whenencapsulated with BSA/Dextran mixtures, typically exhibit in Example 1Example 1 after 15 minutes exposure to 4 M HCL. Briefly, to performdissolution, 100 uL of 0.1 mg/mL (in terms of iron content) of theparticle suspension was incubated with 200 uL of 6 M hydrochloric acidand aliquots of this mixture were removed at various time intervals andassayed for the presence of elemental (or dissolved) iron bycomplexation with potassium thiocyanate as a colorimetric endpointreadout. Table 1, below, shows the acid dissolution behavior of all ofthese aforementioned silanized magnetite clusters.

TABLE 1 Percent dissolution of Iron oxide as a function of acid exposuretime for various silanized magnetite clusters APS + Silicate silanizedTime in 4M EGTA APS only Hydroxymethyl- Hydrochloric Silanized peptizedsilanized silanized acid Magnetite magnetite magnetite magnetitemagnetite (minutes) Superclusters clusters clusters clusters clusters 543.8% 22.8% 21.8% 33.1% 28.1% 10 72.5% 33.7% 38.8% 47.1% 49.1% 15 90.0%48.4% 55.0% 64.9% 68.9% 30  100% 77.0%  100% 89.5%  100% 45  100%  100% 100%  100%  100%

The data in Table 1, above, show that the silanization methods describedherein indeed provide protection against acid dissolution and also serveto provide highly cross-linked silane molecules on the surface of themagnetic particles. For instance, at the 15 minute time point, 90% ofthe (bare) magnetite superclusters were dissolved by acid compared toonly 50% to 70% of the silanized magnetite clusters.

FIG. 1 shows the results of an acid dissolution study performed on asilanized nanomagnetic particle pre- and post-sonication. This studyshows that the silane (glass) coating remained intact on thenanomagnetic particle surface after the second round of high powersonication as described in Example 1, above.

Nanomagnetic particles produced without a primary glass coating aretypically not stable in biological fluids such as plasma and wholeblood, and, furthermore, they are prone to aggregation even in solutionsof low ionic strength. Such protective functionalities (e.g., stability,reduced aggregation) provided by the silanization processes describedherein significantly contribute to the practical utility of the targetednanomagnetic particles claimed in this patent in biological researchefforts as compared to other magnetic nanoparticles.

3. Derivitization, Processing, and Cell Labeling Efficacy of the 70 nmBSA/Dextran-Coated Silanized Nanomagnetic Particle by-Products

The first high-field magnetic wash supernatant from Example 1, above,the magnetic particles in which had a hydrodynamic size of about 70 nm,was first subjected to HGMS purification using a commercially availableHGMS ‘XS’ column (Catalogue#130-041-202; Miltenyi Biotec, San Diego,Calif.), which is packed with small ferromagnetic beads in order toremove the excess coating reagents. The ‘XS’ column was positioned in auniform magnetic field created by positioning a 2 inch×1 inch×0.25 inchthick ‘North’ face and an identically sized ‘South’ face magnet againsteach other. The ‘XS’ column/magnetic assembly was attached to aperistaltic pump to facilitate rapid automated processing of thenanomagnetic particles.

30 mL (12.5 mg iron) of the first high-field magnetic wash supernatantwas HGMS purified into a low-ionic strength HEPES pH7.5 buffer. Afterremoval of the ‘XS’ column from the uniform magnetic field andresuspension with 3 mL of HEPES pH7.5 buffer, about 90% of the particleswere recovered based on iron content. These HGMS-purified nanomagneticparticles had a hydrodynamic diameter of 73 nm and were then conjugatedto a rat anti-mouse CD4 antibody (Catalogue#100506; BioLegend Inc., SanDiego, Calif.) using heterobifunctional coupling chemistry. Briefly, theHGMS-purified 73 nm BSA/Dextran-coated nanomagnetic particles wereactivated with a SMCC cross-linker (Catalogue# S1534; ThermoFisherScientific, San Diego, Calif.) and conjugated to the rat anti-mouse CD4antibody which had been thiolated using 2-Iminothiolane(Catalogue#26101; ThermoFisher Scientific, San Diego, Calif.). The finalhydrodynamic size of these antibody-conjugated nanomagnetic particleswas measured to be 83 nm. Although not thoroughly optimized, when thisconjugated particle was used for targeting mouse CD4⁺ cells fromsplenocyte cell suspensions in conjunction with ‘MS’-type HGMS columns(Catalogue#130-042-201; Miltenyi Biotec Inc., Auburn, Calif.), puritiesand yields in excess of 90% were obtained as measured by flow cytometrywith appropriate fluorescently labeled antibodies (FACSCalibur withCellQuest software; BD Biosciences, San Diego, Calif.).

These results show that these smaller particles, as compared to thelarger ones described elsewhere herein, can also be effectivelyconjugated and utilized for isolation of biological materials.

4. Colloidal Stability of Streptavidin-Conjugated Nanomagnetic Particles

A 115 nm diameter BSA/Dextran-coated nanomagnetic particle producedaccording to Example 1, above, was conjugated covalently to Streptavidinas per the methods described in Example 3, above, to produce 135 nmdiameter Streptavidin-conjugated nanomagnetic particles. Particle sizemeasurements were carried out at various time points after resuspendingand storing the nanoparticles in a high ionic strength solution (1 MNaCl) at room temperature. Control size measurements were carried out onthe same nanoparticles in their normal storage buffer, which was alow-ionic strength buffer supplemented with BSA and sodium azide. Table2, below, shows the results of this study. This study demonstratessignificant resistance to aggregation and enhanced colloidal stabilityof the nanomagnetic particles of this invention.

TABLE 2 Particle Size Stability in 1M Sodium Chloride SIZE @ SIZE @ SIZE@ STORAGE SOLUTION 0 HOURS 1 HOUR 72 HOURS Normal Storage Buffer 135 nm137 nm 137 nm 1M Sodium Chloride 139 nm 139 nm 140 nm

5. Magnetic Separation Efficiency of Nanomagnetic Particles

FIG. 2 shows the magnetic separation efficiency of various nanomagneticparticles, which particles include a 113 nm diameter BSA/Dextran coated,a 127 nm antibody-conjugated particle, and a 130 nmStreptavidin-conjugated particle, the latter two of which are conjugatedusing the method described in Example 3, above. This study was performedusing quadrupole magnetic separator built as described in U.S. Pat. No.5,186,827 and designed to fit standard 12 mm×75 mm disposable laboratorytest-tubes with dilute nanomagnetic particle suspensions containing 25ug/mL iron in a physiological buffer such as an isotonic phosphatebuffered saline solution. Similar strong magnetic separators for usewith test-tubes are available from StemCell Technologies (Part #18000;Vancouver, British Columbia, Canada). FIG. 2 shows that all theseaforementioned nanomagnetic particles separate rapidly andquantitatively within just a few minutes.

Antibody-conjugated commercially available microbeads(Catalogue#130-049-201; Miltenyi Biotec Inc., Auburn, Calif.) having ameasured hydrodynamic diameter of 82 nm were also tested for magneticseparation efficiency in the quadrupole magnetic separator, and theresults shown in FIG. 3. As shown in FIG. 3, these 82 nm microbeads didnot quantitatively separate at all in the quadrupole magnetic separatordescribed above in this example. Instead, only about 40% of thosemagnetic particles could be separated after 30 minutes. These resultsdemonstrate that those types of microbeads are only suitable for usewith HGMS column-based magnetic separation methods. The nanomagneticparticles, however, are suitable for quantitative magneticparticle-based separations in both external-field (dipole, tripole,quadrupole, hexapole type) as well as internal-field (HGMS)-basedmagnetic separators.

This property represents a significant differentiator in terms ofpractical utility of nanomagnetic particles, as no other magneticparticles are presently known to the inventors to function in bothinternal and external types of magnetic separators.

6. Non-Specific Binding of Nanomagnetic Particles to Mammalian Cells

Table 3, below, shows the non-specific binding (NSB) of different lotsof BSA/Dextran-coated nanomagnetic particles synthesized over a 6 monthperiod according to Example 1, above. In this study, mouse splenocytes(1×10⁷ total cells per tube) were incubated for 20 minutes at 4° C. witha relatively large number of nanomagnetic particles (about 2000particles/cell or 2×10¹⁰ total number of particles per sample). Thecell/nanomagnetic particle reaction mixture was then magnetically washedtwice with the aid of a quadrupole magnetic separator using just 5minute magnetic separation times.

The washing steps were performed as follows: the cell suspension wasdiluted to a total volume of 4 mL with an isotonic PBS/BSA/EDTA buffer(5× Phosphate buffered saline (PBS), pH 7.2, 2.5% (w/v) Bovine SerumAlbumin (BSA), and 10 mM EDTA) and the tube placed into aquadrupole-magnetic separator for 5 minutes. The supernatant was thendiscarded by gentle inversion of the magnetic separator or by aspirationwith the aid of a pasteur pipette. The tube was then removed and itscontents resuspended again with 4 mL of the isotonic buffer and placedback into the magnetic separator for another 5 minute separation. Afterthe second aspiration, the cells were centrifuged and the cell pelletwas resuspended with a small volume of isotonic buffer and then analyzedfor the presence of non-specifically collected cells. The magneticallycollected cells were then centrifuged once (5 minutes at 300×g) toremove excess or free nanomagnetic particles. The number of magneticallycollected cells was then counted using an automated cell counter(Cellometered using an Nexcelom Bioscience, Lawrence, Mass.) that,together with the starting number of cells, enabled calculation of thepercentage of cells that were magnetically selected (which is referredto as non-specific binding). Note that 2×10¹⁰ particles is equivalent toa mass of about 40 ug of iron. More typically, for the efficientisolation of cells in high purity and yield, only about 10 ug to 20 ugof nanomagnetic particles (in terms of iron weight) need to be added fora sample containing 1×10⁷ total cells.

These non-specific binding experiments were also repeated using highgradient magnetic separation (HGMS) columns (Part #130-042-201;MS-Columns; Miltenyi Biotec Inc., Auburn, Calif.) in place of thetest-tube quadrupole magnetic separator (see Table 3, below). Due to thevery high magnetic field gradients generated in such HGMS columnseparators, the nanomagnetic particle-to-cell ratio was drasticallyreduced to about 20 particles per 1 cell, or about 2×10⁸ particles persample. It was discovered that particle-to-cell ratios from 10:1 to 50:1provide optimal target cell yields and purities (see Tables 5 and 6,below).

These studies were performed with a commonly used standard, cellcompatible buffer (PBS) supplemented with 0.5 wt % BSA, 2 mM EDTA, and0.1 wt % Casein and adjusted to pH 7.2.

TABLE 3 Particle Magnetic % Non- Particle Diameter Separation SpecificLot # (nm) Method Binding MAG05 110 Quadrupole 1.1 MAG06 113 Quadrupole1.5 MAG07 110 Quadrupole 1.9 MAG08 105 Quadrupole 1.1 MAG09 114Quadrupole 1.4 MAG10 114 Quadrupole 1.1 MAG05 110 MS Column 1.1 MAG06113 MS Column 1.0 MAG07 110 MS Column 1.0 MAG08 105 MS Column 1.2 MAG09114 MS Column 0.8 MAG10 114 MS Column 1.1

The results in Table 3, above, show that the non-specific binding (NSB)of such nanomagnetic particles is extremely low, making it possible toattain target cell purities of up to about 99%. Most, if not all,currently available commercial magnetic particles cannot attain such lowlevels of NSB.

7. Specific Binding and Capture of Mammalian Cells Using a QuadrupoleMagnetic Separator Compared to HGMS Separators

Table 4, below, shows the titration results of a 127 nm diameter ratanti-mouse CD4 antibody- (Clone RM4-5; catalogue#100506; BioLegend Inc.,San Diego, Calif.) conjugated nanomagnetic particle (prepared asdescribed in Example 3, above) with mouse splenocytes. This titrationwas performed using particle-to-cell ratios from 500:1 to 1500:1. Theprotocol used was essentially identical to that described above inExample 6, above, except that after removal of excess nanomagneticparticles, an additional incubation with appropriatefluorochrome-conjugated antibodies (for phenotyping purposes) wascarried out and the cells analyzed on a flow cytometer (FACSCalibur withCellQuest software; BD Biosciences, San Diego, Calif.) to determine thepercent purity of the magnetically selected cells.

TABLE 4 Particle-to-Cell Ratio % Purity % Yield  500:1 93.0 91.2  750:191.6 90.2 1000:1 92.6 92.9 1200:1 92.1 95.1 1500:1 89.3 90.7

This study clearly illustrates the biomedical utility of thenanomagnetic particles of this invention for isolating target cells ofinterest in high yield and purity for further interrogation.

Table 5, below, shows the results of a similar titration study doneusing the same 127 nm diameter rat anti-mouse CD4 antibody-conjugatednanomagnetic particle with mouse splenocytes; however, in this study,HGMS columns were used for performing the magnetic wash steps. Asdescribed to earlier (in Example 6, above), lower particle-to-cellratios, from 5:1 to 50:1, were used in this HGMS based study.

Table 6, below, shows the results of a similar titration study done alsousing HGMS columns but with a 129 nm diameter rat anti-mouse CD19antibody- (Clone 6D5; catalogue#115502; BioLegend Inc., San Diego,Calif.92121) conjugated nanomagnetic particle (prepared as described inExample 3, above) in order to demonstrate the versatility of thenanomagnetic particles of the invention in HGMS-based cell isolationprotocols.

TABLE 5 Particle-to-Cell Ratio (rat anti-mouse CD4) % Purity % Yield 5:1 90.1 55.9 10:1 92.5 84.9 20:1 88.9 95.8 30:1 84.5 98.6 40:1 84.299.1 50:1 83.0 99.2

TABLE 6 Particle-to-Cell Ratio (rat anti-mouse CD19) % Purity % Yield10:1 98.1 82.1 20:1 97.8 96.5 30:1 97.4 98.0 40:1 96.7 98.4 50:1 96.697.9

Both of these studies yielded excellent results for the purity and yieldof the magnetically (purified) captured target cells across a relativelywide range of particle to cell ratios.

A commercially available magnetic particle was measured to have ahydrodynamic diameter of 170 nm and was also titrated as described inthis example, with the results being shown in Table 7, below.

TABLE 7 Particle-to-Cell Ratio % Purity % Yield 10:1 93.7 88.7 15:1 91.060.0 20:1 95.2 62.0 30:1 93.9 37.0

This commercially available magnetic particle did not exhibit asufficiently wide particle-to-cell usage ratio such that reliable andreproducible results could be obtained, therefore indicating that suchconventional magnetic particles are not compatible for use withHGMS-type magnetic separation methods. The rapid loss of yield upontitration with those magnetic particles was most likely due toentrapment of the relatively large sized magnetic particles in themetallic (or ferromagnetic) matrix in the HGMS column, leading toinefficient recoveries of the magnetically labeled cells retained on thede-magnetized HGMS column. As those in the art will appreciate, suchconventional magnetic particles can only be practically used with strongexternal-field magnetic separators such as the quadrupole-type magneticseparators used in the studies described above.

8. Stability of the Nanomagnetic Particles Produced According toExamples 1 and 3, Above

To assess the long-term stability of the nanomagnetic particlesdescribed above, both Streptavidin- and rat anti-mouse CD19antibody-conjugated particles were prepared according to Examples 1 and3, above. Multiple small aliquots of these particles were then stored atthree different temperatures (4° C., 25° C., and 37° C.) and magneticcell separation tests were performed at various time points over thecourse of two months in order to monitor the overall biostability ofthese nanomagnetic particles. BioLegend. MojoSort™ Mouse CD4 T CellIsolation Kit (Catalogue#480005) is a negative selection test that usesStreptavidin nanomagnetic particles in conjunction with a cocktail ofbiotinylated antibodies in order to magnetically select all cells thatare CD4 negative. Additionally, BioLegend MojoSort™ Buffer and MojoSort™Magnet were used in the execution of the cell selection protocolsdescribed in this example. The “untouched” cells or supernatant from themagnetic separation step contained the desired CD4-positive cells. These“untouched” cells were then analyzed on a flow cytometer (FACSCaliburwith CellQuest software; BD Biosciences, San Diego, Calif.) in order todetermine the purity and yield of the targeted CD4-positive cells.Similar analyses were also performed using BioLegend DieMojoSort™ MouseCD19 Nanobeads (BioLegend, Catalogue#480001), which are rat anti-mouseCD19 antibody-conjugated nanomagnetic particles used to positivelyselect CD19-positive cells.

FIGS. 4 and 5 show the purity and yield, respectively, of CD4 positivecells that were negatively selected using Streptavidin nanomagneticparticles and appropriate biotinylated antibodies that were stored atvarious temperatures and tested for cell separation performance over thecourse of two months.

Similarly, FIGS. 6 and 7 show the purity and yield, respectively, of ratanti-mouse CD19 antibody conjugated nanomagnetic particles that werestored at various temperatures and tested for cell separationperformance over the course of two months. Note that a noncross-reacting, fluorescently labeled B-cell-specific antibody calledCD45R/B220 (Catalogue#103223; BioLegend Inc., San Diego, Calif.) wasused to identify the magnetically selected B cells.

The results of these stability studies clearly demonstrate the excellentbiostability of such nanomagnetic particles. The fact that both sets ofnanomagnetic particles used in these studies have at least 30 or moredays of biostability at an elevated temperature of 37° C., which can beextrapolated to upwards of more than 4 years of biostability at 4° C.,can be attributed to the nanomagnetic particle composition and synthesisdesigns. In contrast, conventional magnetic particles ranging in sizefrom 80 nm to 1000 nm have been reported to have shelf-lives orbiostability in the range of a few months to about 20 months even whenrefrigerated at 4° C. As such, conventional magnetic particles are lesspreferred but can still be used in various embodiments of themagnetibuoyant separations described herein.

9. Comparison of Particle Size Distributions

The particle size distributions of various conventional, commerciallyavailable magnetic particles that are highly utilized in the targetedcell separations market were measured and compared to those producedusing Example 1, above. These measurements were made using dynamic lightscattering (Malvern Nano-S ZetaSizer; Westborough, Mass.), and thepercentage of particles in various ‘size-bins’ was plotted as a functionof actual particle size, as shown in FIG. 8. Hydrodynamic diameters aremeasured on a Malvern Nano-S ZetaSizer instrument that uses theprinciples of ‘dynamic light scattering’ whereby particles areilluminated with a laser and the scattered light analyzed for intensityfluctuations. The nanomagnetic particles (labeled as “BioLegend” in FIG.8) had a hydrodynamic diameter of about 130 nm and relativelyinsignificant numbers of particles greater than about 300 nm in diameter(an important criterion in order for magnetic particles to performequally well in both ‘external-field’ and ‘internal-field’ basedmagnetic separators). Note that the particles labeled “ConventionalParticle ‘A” in FIG. 8 had a hydrodynamic diameter of about 82 nm andtherefore would only be suitable for use with Internal-field’ generatingor HGMS columns (see FIG. 3, above, also).

10. Transmission Electron Microscopy of Cells Selected Using HGMS

In this study, cells were magnetically selected by HGMS using bothcommercially HGMS compatible magnetic particles (FIGS. 9A, 9C, 9E) andtargeted nanomagnetic particles of the present invention (FIGS. 9B, 9D,9F). The representative electron micrographs shown in FIG. 9 wereproduced using 55 nm cryosections of the magnetically selected cells andimaged on a Transmission Electron Microscope. A single cell suspensionfrom C57BL/6 mouse spleen was prepared to isolate CD19+ B cells usingthe MojoSortred light anaNanobeads (BioLegend, CA) and commercial mouseCD19 MicroBeads (Miltenyi, Germany) followed by BioLegend and Miltenyirecommended protocol. Isolated CD19 cell purity (97% from BioLegend,94.9% from Miltenyi) was identified by staining of the resulting cellswith CD45R/B220 (clone RA3-6B2) PE and analysis by flow cytometry. Thenthe cells were centrifuged and the cell pellets were resuspended in amodified Karnovsky's fixative (2.5% glutaraldehyde and 2%paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4) for 4hours. Then the preparation was post-fixed in 1% osmium tetroxide in0.15 M cacodylate buffer for 1 hour and stained en bloc in 2% uranylacetate for 1 hour. Samples were then dehydrated in ethanol, embedded inDurcupan epoxy resin (Sigma-Aldrich), sectioned at 50 to 60 nm on aLeica UCT ultramicrotome, and picked up on formvar and carbon-coatedcopper grids. Sections were stained with 2% uranyl acetate for 5 minutesand Sato's lead stain for 1 minute. Grids were then viewed on a JEOL1200EX II (JEOL, Peabody, Mass.) transmission electron microscope andphotographed using a Gatan digital camera (Gatan, Pleasanton, Calif.),or viewed using a Tecnai G2 Spirit BioTWIN transmission electronmicroscope equipped with an Eagle 4 k HS digital camera (FEI, Hilsboro,Oreg.).

Similarly low numbers of the targeted nanomagnetic particles compared tothose of conventional labeled magnetic particles were observed across 40images from each sample type. These electron micrographs clearly showthat far fewer of targeted nanomagnetic particles are bound to thetarget cells than in the micrographs showing cells bound by conventionallabeled magnetic particles. The arrows in these micrographs mark thelocation of visualizable magnetic particles on the surface of thesecells. This (the ability to mediate magnetic separation with very fewnanomagnetic particles per cell) is a very important attribute of thetargeted nanomagnetic particles because such magnetically selected cellsare essentially in a “native” or “untouched” state with very little, ifany, perturbation of the cells. This allows the cells to be captured ina biologically intact and responsive state (see Example 12).

11. Nanomagnetic Particle Lyophilization Studies

Mouse anti-CD19 conjugated nanobeads (2×10⁸ total particles) and SAvconjugated nanobeads (2×10⁸ total particles) produced according toExamples 3 and 4, above, respectively, were suspended in varioussupplemented solutions and subjected to a 3 day lyophilization (Lyo)cycle on a Genesis Pilot Lyophilizer (SP Scientific). Specifically,particle suspensions contained in silanized glass vials were frozen downto ˜46° C., then to ˜80° C. for 3 hours and back to ˜46° C. and kept ina sealed vacuum chamber for 3 days. Thereafter, the temperature wasraised to 22° C. The lyophilized nanomagnetic particles were thenreconstituted with PBS and tested for performance using both theMojoSort™ Mouse CD19 Nanobeads (BioLegend Inc., San Diego, Calif.;catalogue #480001) and the MojoSort™ Mouse CD4 T Cell Isolation Kits(BioLegend Inc., San Diego, Calif.; catalogue #480005). The resultsshown in Tables 8 and Table 9, respectively, below.

TABLE 8 Mouse CD19 positive selection purity and yield by usingreconstituted lyophilized (lyo) CD19 nanobeads Particles Purity (%)Yield (%) Non Lyophilized 6D5 particle (Control) 97.7 82 6D5 nanobeadsin Storage Buffer (Lyo) 97.1 72 in 1% BSA (Lyo) 96.9 88 in 1% Dextran(Lyo) 96.9 88.2 in 2% Sucrose (Lyo) 97 89.2 in 1% Dextran + 1% Sucrose(Lyo) 96.7 90.8

TABLE 9 Mouse CD4 negative selection purity and yield by usingreconstituted lyophilized (lyo) SAv particles Particles Purity (%) Yield(%) Non lyophilized SAv (Control) 95.4 90.0 in 1% BSA (Lyo) 92.8 92.9 in1% Dextran (Lyo) 96 88.1 in 2% Sucrose (Lyo) 96 87.0 in 1% Dextran + 1%Sucrose (Lyo) 96.2 87.6

These lyophilized and reconstituted nanomagnetic beads show excellentretention of bioactivity, indicating that lyophilization facilitatesextended storage/stability of targeted nanomagnetic beads for very longperiods of time.

12. Functional Studies of Magnetically Selected Cells

Magnetically selected cells are often used for downstream processingsuch as gene/protein/RNA profiling; however, many if not most ofcommercially available magnetic particles have a toxic effect on cells,Therefore, it is quite challenging to obtain live or viable cells withmagnetic particles attached to them for further studies/probing. In thisstudy, both a targeted nanomagnetic particle species and a widely usedcommercially available magnetic particle species conjugated to anantibody against the mouse CD4 antigen were tested side-by-side for cellfunctionality after the target CD4⁺ cells were magnetically isolated.

Briefly, a rat anti-mouse CD4 antibody (Clone RM4-5; catalogue#100506;BioLegend Inc., San Diego, Calif.) conjugated nanomagnetic particle(prepared as described in Example 3, above) was tested alongsideanti-CD4 (Clone L3T4; Catalogue#130-049-201; Miltenyi Biotec Inc.,Auburn, Calif.) conjugated microbeads using HGMS columns(Catalogue#130-042-201; Miltenyi Biotec Inc., Auburn, Calif.). Theanti-CD4-conjugated nanomagnetic particles of the invention had ahydrodynamic diameter of 127 nm whereas the L3T4-conjugated microbeadshad a hydrodynamic diameter of 82 nm. Table 10, below, shows the purityand yield of the isolated CD4⁺ cells from both types of these magneticparticles when used for isolating CD4⁺ cells from a mouse spleenaccording to the manufacturer's instructions.

TABLE 10 Type of nanomagnetic particle used % PURITY % YIELD BioLegendanti-CD4 nanobeads 92.4 65 MACS anti-CD4 MicroBeads 91.5 67

After magnetic isolation of the CD4⁺ cells, equal amounts of CD4⁺ cells(lx 10⁶ cells) from both isolation methods were seeded into 96-wellmicroplates coated with mouse anti-CD3 (Clone 17A2; catalogue#100201;BioLegend Inc., San Diego, Calif.) antibody in varying concentrationsand supplemented with 1 ug/mL soluble mouse anti-CD28 (Clone 37.51;catalogue#102101; BioLegend Inc., San Diego, Calif.) and incubated for 3days at 37° C. Next, a solution of the fluorescent redox markerresazurin (catalogue# TOX8-1KT; SIGMA-ALDRICH; St. Louis, Mo.), whichmeasures the metabolic activity of living cells, was added into thewells at a 10% volume ratio and the relative fluorescence intensity wasmeasured after a 7 hour incubation using a SPECTRAmax Gemini XPSfluorescence microplate reader (Molecular Devices, Sunnyvale, Calif.). Aplot of the relative fluorescence units (RFU) versus the concentrationof the anti-mouse CD3 antibody used for coating the microwells is shownin FIG. 10. Note that in FIG. 10, the higher the fluorescence intensity,the higher the number of living cells.

The results of this functional cell assay clearly demonstrate thatnanomagnetic particles do not have a significant toxicological effect onmagnetically selected cells even though such nanomagnetic particles arelarger than the tested commercial magnetic microbeads.

13. Combined Use of Microbubbles in Conjunction with NanomagneticParticles for Cell Isolation

Micro-sized buoyant bubbles are hollow (or air- or specific gas-filled)micron-sized spheres that are commercially available with functionalizedsurfaces or coated ligands for targeting moieties of interest.Commercially available examples that could be conjugated withcell-specific ligands (e.g., cell antigen specific antibodies) and usedto isolate specific cell populations include the gas-filled phospholipidmicrobubbles formerly available from Targeson (San Diego, Calif.) andglass Buoyant Microbubbles from Akadeum Life Sciences (Ann Arbor,Mich.). Examples described in the research literature include theperfluorocarbon microbubbles of Shi, et al., Methods, 64, 102 (2013),glass microbubbles of Hsu, et al., Technology (Singapore World Science),3, 38 (2014), albumin microbubbles of Liou, et al., PLoS One, 20, 10(2015), and gas-filled immune-microbubbles of Shi, et al., PLoS One, 8,1 (2013). Examples of patent literature describing the use ofmicrobubble-based systems for isolation of analytes or cells includeU.S. patent and published patent application U.S. Pat. Nos. 5,116,724,5,246,829, 8,835,186, US 20030104359, US 20070036722, and US20110236884. These examples illustrate the value of using abuoyancy-based system for the specific isolation of target cells andanalytes. Yet, prior to this invention, none have combined abuoyancy-based system with magnetic particles to provide faster, moreefficient, and more effective enrichment and isolation of the desiredbiomolecular target (e.g., a particular cell type) or of multipletargets.

In this example, a unique method is described wherein both targetedmicrobubbles, of any composition, and targeted nanomagnetic particles,of any composition, can be used sequentially and/or simultaneously toobtain one, two, or three cell populations of interest more quicklyand/or at higher purity than with magnetic particle or microbubbletechniques alone. A combination of magnetic and buoyant isolation, or“magnetibuoyant”, procedures will allow difficult separations to beachieved very efficiently. Such “magnetibuoyant” methods of cellisolation significantly reduce the time and resources required toisolate different cells of interest, and the populations can be obtainedat very high purities. Targeted magnetic nanoparticles are particularlywell-suited for this application due to their high stability in variousfluids, small size, higher magnetic responsiveness property, ability toseparate cells at lower particle to cell ratios as compared to othermagnetic particles and capacity to respond more quickly and completelyto magnetic fields as compared to other magnetic particles. Theseadvantages have not previously been realized and/or commercialized. FIG.11 illustrates the general principle for isolating two distinctpopulations, and FIG. 12 illustrates the same general principle forisolating three distinct populations.

Considering FIG. 11, if a mixture of different cell types (A, B, C, D)containing two (A and B) desired, or wanted, subpopulations are combinedin a reaction mixture with microbubbles targeted to one desired celltype (A) and with magnetic particles targeted to a second desired type(B), then allowing the first set of cells (A) to float to the surfacewhile the second set of cells (B) is drawn to a strong magnetic field(such as the quadrupole magnetic separator described in Example 5,above), this will cause the magnetized target cells to be separated atright angles to the levitation direction of the microbubble-targetedcells. In this manner both populations of cells can be isolated after asingle separation step and can be harvested individually for further usefrom the same initial reaction mixture. In this simple example both ofthe different cell populations (A and B) may be desired for further use,and can be easily harvested. Alternatively, one population may beunwanted cells that will be discarded with, for example, the intent ofremoving them as potential contaminants of the second isolatedpopulation.

Considering FIG. 12, the third “remainder” population (in this example,cell types C and D), i.e., those not targeted by either the microbubblesor the magnetic particles, may also be harvested for further use, ifdesired, since that population can also be separately retained as thetwo targeted populations (A and B) are harvested. For example, after acombined buoyant microbubble-labeling, magnetic nanoparticle-labelingand sedimentation step, the buoyant cells (A) could be harvested bypipetting, pouring or aspiration, followed by removal of the remainingcell buffer and harvesting of the sedimented population (C and D),followed by release and harvest of the magnetic population (B) from thewalls of the tube in fresh cell buffer. This procedure would result inthree enriched populations after a single labeling and preparation step.

As background information to show that microbubbles can be effective atenriching and isolating cell populations, two examples are provided toillustrate how microbubbles alone can levitate cell populations fromcomplex mixtures of different origin. FIG. 13 illustrates the generalprinciple of using only microbubbles conjugated with an anti-mouse CD19antibody to float and positively isolate mouse CD19⁺ cells, the wantedcell population. In this example, biotinylated rat anti-mouse CD19(Clone 6D5; catalogue #115503; BioLegend Inc., San Diego, Calif.)conjugated to streptavidin phospholipid microbubbles are incubated withmouse splenocytes for 15 minutes at 4° C. in a small eppendorf tube on arotator (see (a) in FIG. 13). The cell suspension is then transferredinto a test tube and diluted up to a total volume of 4 mL with isotoniccell buffer and centrifuged for 5 minutes at 300×g (see (b), FIG. 13).The floating cells are gently poured or aspirated and transferred into anew test-tube (c). The phospholipid microbubbles attached to the cellsvia antibody bound to CD19 are then burst by transfer to a microsyringeand application of mild pressure via the syringe plunger. Destruction ofthe bubbles eliminates the buoyancy property and allows for subsequentstaining of the cells for flow cytometric analysis. The cells are thenstained with a fluorescent CD45R/B220 antibody conjugate (phycoerythrinconjugated rat anti-mouse/human CD45R/B220; catalogue#103207; BioLegendInc., San Diego, Calif.), and the collected cells analyzed on a flowcytometer (FACSCalibur with CellQuest software; BD Biosciences, SanDiego, Calif.). Table 11, below, shows the purity and yield of the mouseCD19⁺ target cells obtained using such antibody conjugated microbubbles.This shows a nearly two-fold enrichment of the CD19⁺ population in theharvested buoyant population, from 55% to nearly 99% pure CD19⁺ cells.

TABLE 11 Pre Isolation Post Isolation Purity 55% 98.6% Yield 100% 92.4%

In the second example using microbubbles alone, the bubbles were used tofloat and eliminate unwanted cells from the sample so that the remainingnon-buoyant population contains an enriched fraction of the wantedcells. FIG. 14A illustrates the general principle used where a cocktailof different antibodies attached to microbubbles are used to float mostof the undesired cell types (A and B) so that the wanted cells (D) areenriched in the non-buoyant population that can also be harvestedthrough standard sedimentation techniques. A bone marrow suspension wasprepared from C57BL/6 mice according to BioLegend's “Preparation ofMouse and Rat Tissue Single Cell Suspension” Standard OperatingProcedure. The cells were suspended to 1×10⁸/ml and 100 ul of cells weretransferred into a 1.5 ml Eppendorf tube. Then the cells were incubatedwith 10 ul of MojoSort™ Mouse Hematopoietic Progenitor Cell IsolationCocktail (BioLegend, Catalogue #480004) for 15 minutes on ice to targetthe majority of non-hematopoietic progenitor cells with biotinylatedantibodies, followed by two washes to remove extra biotinylatedantibody. The Mouse Hematopoietic Progenitor Cell Isolation Cocktailcontains antibodies recognizing several targets on mouse bone marrowcells (unwanted cell types) that are not mouse hematopoietic progenitorcells (wanted cells). The cell pellet was re-suspended with 100 ul ofPBS and the tube was held at a 45° angle and 1×10⁷ StreptavidinMicrobubbles (Akadeum Life Science, Ann Arbor, Mich., Catalogue # SA01)were slowly added to the tube and gently triturated for 1 minute,followed by 360° rotation for another 10 minutes at room temperature.Then the cell-microbubble mixture was transferred into a 15 ml tube andPBS was added up to 14 ml followed by centrifugation for 5 minutes at300×g. With this procedure the “non-hematopoietic progenitor” cells willrise and the desired hematopoietic progenitor cells will form a pellet.After aspirating the floating cells and medium, the cell pellet wasresuspended with 100 ul of PBS and incubated with 1 ul of PE anti-mouseLy-6A/E (Sca-1) (clone D7) (BioLegend, San Diego, Calif., Catalogue#108108) and APC anti-mouse CD117 (c-Kit) (clone 2B8) (BioLegend, SanDiego, Calif., Catalogue #105812) according to BioLegend “DirectStandard Staining of Mouse and Rat Leukocytes” Standard OperatingProcedure to label the hematopoietic stem cells. Then the cells wereanalyzed by flow cytometry (FACSCalibur with Cell Quest software; BDBioscience, San Diego, Calif.) according to BioLegend cell acquisitionStandard Operating Procedure, and data were analyzed with FlowJoanalysis software (FlowJo, LLC, Ashland, Oreg.). Typical flow cytometryresults are shown in FIGS. 14B and 14C in which quadrant 2 (Q2) of thecytograms shows the frequency of CD117⁺/Sca⁻1⁺ cells in the populationeither before or after pre-enrichment with multiple antibody conjugatedmicrobubbles. For comparison, similar enrichments using magneticnanoparticles to remove the unwanted cells (BioLegend kit #480003) werealso performed. Enrichment results for the two methods are compared inTable 12. The results in Table 12, below, show that rare hematopoieticstem cells can be enriched six to seven fold by using microbubblesconjugated to multiple antibodies of different specificities to removeundesired contaminating cells. This is similar to the degree enrichmentseen using magnetic nanoparticles.

TABLE 12 CD117⁺/Sca-1⁺ CD117⁺/Sca-1⁺ Fold frequency frequency enrichmentbefore after of CD117⁺/ pre-enrichment pre-enrichment Sca-1⁺ cells HSCpre-enrichment by 0.2-0.5% 1.2-3.5 6-7 microbubbles HSC pre-enrichmentby 1.4-4.5 7-9 magnetic beads

14. Simultaneous Isolation of Mouse CD4⁺ Cells and CD19⁺ Cells by UsingStreptavidin-Microbubbles and Anti-Mouse CD19 Magnetic Nanoparticles

In biological research it is often desirable to isolate two or morespecific subpopulations of cells, such as CD19⁺ B lymphocytes and CD4⁺ Tlymphocytes, from a complex cell mixture, such as the mouse spleen,which can contain many different subpopulations of B cells, T cells,monocytes, and dendritic cells, for example. Current work flows requireperforming separate procedures on separate starting mixtures to isolatethe different subpopulations. It would be advantageous in terms ofreagents, time and tissue costs to be able to isolate more than onepopulation from a single complex starting mixture. The exampleillustrated in FIG. 15A shows such savings enabled by the invention. InFIGS. 15B-15D CD4⁺ cells and CD19⁺ cells, both from the same complexmouse splenocyte preparation, are individually isolated during the samework flow.

Mouse splenocytes were prepared from a C57BL/6 mouse. The splenocyteswere suspended at 1×10⁸/ml in a suitable buffer and 100 μl of thesplenocytes was then transferred into 1.5 ml Eppendorf tube. Then thecells were incubated with 2.5 μg of biotinylated anti-mouse CD4 (cloneRM4-5) (BioLegend, San Diego, Calif., Catalogue #100508) for 15 minuteson ice followed by two washes to remove unbound biotin antibody. Cellswere resuspended in 100 μl PBS, pH 7.4 (Thermo Fisher, San Diego,Calif., Catalogue #10010023) and incubated with 10 μl of magneticMojoSort™→ Mouse CD19 Nanobeads (BioLegend, San Diego, Calif., Catalogue#480002), nanomagnetic particles conjugated with anti mouse-CD19monoclonal antibodies, on ice for 14 minutes. Then the tube was held ata 45° angle and 1×10⁷ Steptavidin Microbubbles (streptavidin-coatedmicrobubbles) Akadeum Life Science, Ann Arbor, Mich., Catalogue #SA01)were slowly added and triturated for 1 minute. Thestreptavidin-conjugated microbubbles attached to the previously boundbiotinylated anti mouse-CD4 antibody. Then PBS was added up to 3 mltotal volume and the tube was placed in a MojoSort™ Magnet (BioLegend,San Diego, Calif., catalogue #480019) for three iterative separationsteps. With this procedure the magnetic nanoparticle-bound cellsattached to the magnet and the microbubble-bound cells rose. After eachseparation step, the floating cell-microbubble mixture was poured intoanother new tube. Then the harvested cell-microbubble and cell-magneticnanoparticle containing tubes were resuspended in PBS and centrifuged at300×g for 5 minutes. With this procedure the CD4⁺ cells rose and CD19⁺cells formed a pellet. The CD19-enriched cell pellet was then stainedwith 1 μl of PE anti-mouse CD4 (clone RM4-4) (BioLeged, San Diego,Calif., Catalogue #116006) and FITC anti-mouse CD19 (clone 6D5)(BioLegend, San Diego, Calif., Catalogue #115506). and centrifuged at300×g for 5 minutes. The cells were acquired by flow cytometry(FACSCalibur with Cell Quest software; BD Bioscience, San Diego,Calif.). It was not possible to analyze by conventional flow cytometrythe CD4-enriched floating cell population obtained with this form ofantibody-conjugated glass microbubble due to obstruction of sample flowin the cytometer fluidics by the retained microbubbles. Instead, thepercentage of the CD4⁺ cells depleted from the non-bouyant fraction wasused to calculate the percentage of cells harvested into the buoyantfraction.

The results of the experiment (Table 13, below) show that this protocolfor simultaneously enriching two populations from a single complexmixture can rapidly, efficiently, and effectively provide bothsubpopulations in high purity and yield. The purity of CD19⁺ cells andCD4⁺ cells isolated by the simultaneous magnetibuoyant method is similarto those by separate magnetic nanoparticle methods. Streptavidinmicrobubbles conjugated with biotinylated antibody can be generated in astable stock format so that the stock could be used instantaneously,without a conjugation step, and substantially reduce the cell isolationtime. In alternative and improved product formats the wanted cells couldbe released from the microbubbles by a variety methods such as by usingreversible heterobifunctional crosslinking agents, competitive analoguedisplacement techniques such as biotin/desthiobitin, as well as bycollapsing phospholipid microbubbles by gentle mechanical means.

TABLE 13 CD4 CD4 CD19 CD19 purity isolated % purity yield Simultaneouslyisolate CD4 and Not 90.6% 98.3 77.03% CD19 positive cells Done CD4positive selection with anti- 92-95% 90-94% NA NA mouse CD4 nanobeadsCD19 positive selection with NA NA 95-98% 95-98% anti-mouse CD19nanobeads

15. Magnetibuoyant Method for Isolating Human CD4⁺ Cells at Very HighPurities

Commercially available methods for isolating rare cells (i.e., cellssuch as stem cells, circulating tumor cells, fetal cells, endothelialcells, etc.) are magnetic particle-based, two-step protocols where anegative depletion step is carried out first to remove unwanted cellsfollowed by multiple washing steps in an effort to removenon-specifically bound magnetic particles. Then a positive selectionstep, again with multiple washes, is performed to capture rare cells.The direct positive selection of rare cells has only limited success dueto non-specific binding of the solid-phase materials (i.e., magnetic andnon-magnetic beads), losses due to multiple wash steps, and the immensedifficulty in targeting and binding to rare cells. Furthermore, thestarting cell suspensions often used for direct positive selection ofrare cells are typically very complex mixtures. Any significantmanipulation of the starting or native cell suspension, such asrepetitive washes, has a negative impact on the recovery or yield of anyrare cells present in the sample due to inherent cell losses experiencedat every stage of cell sample processing.

As an example of the use of a magnetibuoyant method of the invention toreduce sample loss and improve workflow efficiency, the method was usedto significantly improve the purity of human CD4⁺ lymphocytes isolatedfrom a peripheral blood mononuclear cell preparation (PBMC). In thisexample the CD4 antigen is also co-expressed on unwanted monocytes atlevels high enough to cause them to be co-isolated with the desired,targeted CD4⁺ lymphocytes. Current methods for isolating human CD4⁺lymphocyte cells from PBMC in high purity requires a pre-enrichment stepto remove contaminating monocytes either with magnetic particles or byadherence to plastic plates, both using multiple time consumingprocedures. This is followed by coating CD4⁺ lymphocytes with antiCD4-conjugated magnetic particles and isolation of CD4⁺/CD14⁻lymphocytes via additional magnetic separation steps. This exampledemonstrates a faster, simpler work flow for obtaining highly pureCD4⁺/CD14⁻ lymphocytes at high yield.

As diagrammed in FIG. 16, a monocyte-specific biotinylated antibodyrecognizing the monocyte marker CD14, clone #63D3 (catalogue #367102;BioLegend Inc., San Diego, Calif.), was conjugated to streptavidinconjugated microbubbles and an anti-CD4-specific antibody, such as clone#SK3 (catalogue #344602; BioLegend Inc., San Diego, Calif.), wasdirectly conjugated to nanomagnetic particles. The PBMCs were thencoated with the two antibody preparations and first subjected to buoyantremoval of the CD14⁺ cells, including the unwanted CD4⁺/CD14⁺ monocytes.This was followed by magnetic isolation of the CD4⁺/CD14⁻ lymphocytepopulation. With magnetibuoyant cell isolation the buoyant CD14⁺/CD4⁺double positive monocytes are lifted away from the CD4⁺ lymphocytes,which were then captured to the walls of the tube with magnetic force athigher purity than without CD14 depletion first. This resulted in asignificant reduction in processing time so that increased throughputcan be realized. Flow cytometric analyses of the PBMC complex mixturebefore and after pre-removal of the contaminating CD4⁺/CD14⁺ monocytepopulation using microbubbles in a continuous workflow is shown in FIG.16, and a summary of the results are shown in Table 14, below. Theresults show (FIGS. 17A-17F) an improvement in the purity of theCD4⁺/CD14⁻ lymphocyte population to nearly 100% purity using this simpleand rapid procedure. The yield of cells in the desired population isequivalent to that obtained without the pre-removal step. These resultsshow utility of the invention for easily obtaining increasingly purepopulations of desired cells characterized by a second marker alsopresent on unwanted cells but not on the desired cell population.

In the two examples of magnetibuoyant separation shown above,biotinylated targeting antibody was used to conjugate thestreptavidin-coated microbubbles so that the principle of improvedseparations could be demonstrated. Direct conjugation of the antibodiesto the microbubbles is also possible and this step would simplify andaccelerate the workflow while retaining the positive attributes of themethod.

TABLE 14 CD14 CD4 CD4 depleted % purity yield CD4 positively selectedwith 75-80% 96-99%   95-97% microbubble pre-depletion of CD14 CD4positively selected without NA 85-89.6% 95-99% microbubble pre-depletionof CD14

Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected below. In addition to theseterms, others are defined elsewhere in the specification, as necessary.Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below (or elsewhere in thespecification). Unless explicitly stated otherwise, or apparent fromcontext, the terms and phrases below (or those defined elsewhere in thespecification) do not exclude the meaning that the term or phrase hasacquired in the art, as these definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Unless otherwise expressly defined below or elsewhere in thespecification, terms of art used in this specification will have theirart-recognized meanings.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, references to “the method” include one or more methods and/orsteps of the type described herein and/or which will become apparent tothose persons skilled in the art upon reading this disclosure and soforth. Similarly, the word or is intended to include “and” unless thecontext clearly indicates otherwise.

The term “about” refers to approximately a +/−10% variation from thestated value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

The term “administering” refers to the placement of a compositionaccording to the invention into a subject by a method or route thatresults in at least partial localization of, for example, theadministered cells, at a desired site. A pharmaceutical composition canbe administered by any appropriate route that results in an effectivetreatment in the subject.

An “analyte” refers to the substance to be detected, which may besuspected of being present in the sample (i.e., the biological sample).The analyte can be any substance for which there exists a naturallyoccurring specific binding partner or for which a specific bindingpartner can be prepared. Thus, an analyte is a substance that can bindto, or be bound by, one or more specific binding partners.

An “antibody” refers to a protein consisting of one or more polypeptidessubstantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes. This term encompasses polyclonal antibodies,monoclonal antibodies, and antigen-binding antibody fragments, as wellas molecules engineered from immunoglobulin gene sequences thatspecifically bind an antigen of interest. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE,respectively. A typical immunoglobulin (antibody) structural unit isknown to comprise a tetramer. Each tetramer is composed of two identicalpairs of polypeptide chains, each pair having one “light” (about 25 kD)and one “heavy” chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms “variable lightchain (VL)” and “variable heavy chain (VH)” refer to these light andheavy chains, respectively.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized antigen-binding antibody fragments produced bydigestion with various peptidases. Thus, for example, pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab′)₂, a dimer of Fab which itself is a light chain joined to VH-CH1by a disulfide bond. The F(ab′)2 may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)₂ dimer into a Fab′ monomer. The Fab′ monomer is essentially a Fabwith part of the hinge region. While various antigen-binding antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments may be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, in the context of the invention the term “antibody” also includesantigen-binding antibody fragments either produced by the modificationof whole antibodies or synthesized de novo using recombinant DNAmethodologies. Antibodies include single chain antibodies (antibodiesthat exist as a single polypeptide chain), single chain Fv antibodies(sFv or scFv), in which a variable heavy and a variable light chain arejoined together (directly or through a peptide linker) to form acontinuous polypeptide. The single chain Fv antibody is a covalentlylinked VH-VL heterodimer that may be expressed from a nucleic acidincluding VH- and VL-encoding sequences either joined directly or joinedby a peptide-encoding linker. While the VH and VL are connected to eachas a single polypeptide chain, the VH and VL domains associatenon-covalently. The scFv antibodies and a number of other structuresconvert the naturally aggregated, but chemically separated, light andheavy polypeptide chains from an antibody V region into a molecule thatfolds into a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart.

An “autoimmune disease” herein is a non-malignant disease or disorderarising from and directed against an individual's own tissues. Examplesof autoimmune diseases or disorders include, but are not limited to,inflammatory responses such as inflammatory skin diseases includingpsoriasis and dermatitis (e.g. atopic dermatitis); systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); respiratory distresssyndrome (including adult respiratory distress syndrome; ARDS);dermatitis; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; allergic conditions such as eczema and asthma andother conditions involving infiltration of T cells and chronicinflammatory responses; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus (e.g. Type I diabetes mellitus or insulin dependent diabetesmellitis); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious anemia (Addison's disease); diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia (including,but not limited to cryoglobinemia or Coombs positive anemia); myastheniagravis; antigen-antibody complex mediated diseases; anti-glomerularbasement membrane disease; antiphospholipid syndrome; allergic neuritis;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia, etc.

A “binding partner” or “member” of a high affinity binding pair is amember of a binding pair, i.e., a pair of molecules wherein one of themolecules binds to the second molecule. Binding partners that bindspecifically are termed “specific binding partners.” A “high affinity”binding pair is one in which the members bind with high affinity. Inaddition to antigen and antibody binding partners commonly used inimmunoassays, other specific binding partners can include biotin andavidin (or streptavidin), carbohydrates and lectins, nucleic acids withcomplementary nucleotide sequences, ligand and receptor molecules,cofactors and enzymes, enzyme inhibitors and enzymes, and the like.Furthermore, specific binding partners can include partner(s) thatis/are analog(s) of the original specific binding partner, for example,an analyte-analog. Immunoreactive specific binding partners includeantigens, antigen fragments, antibodies (monoclonal and polyclonal) andantigen-binding antibody fragments.

A “biological sample” is a sample of biological material taken from apatient or subject, as well as samples taken from tissue culture ortissue culture supernatants or any other source that could contain theanalyte of interest. Biological samples include samples taken frombodily fluids and tissues (e.g., from a biopsy) or tissue preparations(e.g., tissue sections, homogenates, etc.). A “bodily fluid” is anyfluid obtained or derived from a subject suitable for use in accordancewith the invention. Such fluids include whole blood, blood fractionssuch as serum and plasma, urine, sweat, lymph, feces, ascites, seminalfluid, sputum, nipple aspirate, post-operative seroma, wound drainagefluid, saliva, synovial fluid, bone marrow, cerebrospinal fluid, nasalsecretions, amniotic fluid, bronchoalveolar lavage fluid, peripheralblood mononuclear cells, total white blood cells, lymph node cells,spleen cells, and tonsil cells.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung, squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, and various types of head andneck cancer.

The term “cell sample” refers to suspensions or mixtures of cells havingdifferent phenotypes or cell subpopulations in different amounts, forexample, cells that are common in whole blood, peripheral blood,umbilical cord blood, and bone marrow, buffy coat fractions, as well ascell preparations generated by, for example, leukapheresis. Such cellsamples may include, for example, erythrocytes, platelets, andleukocytes, such T-cells, regulatory T-cells, B-cells, NK cells,dendritic cells, monocytes, granulocytes, and/or hematopoietic stemcells.

The term “comprising” or “comprises” is used in reference to articles,compositions, methods, and respective component(s) thereof that areessential to the article, composition, or method, as the case may be,yet open to the inclusion of unspecified elements, whether essential ornot. It is synonymous with “including,” “containing,” “characterizedby,” or like open-ended terms or phrases

The term “consisting essentially of” refers to those elements requiredfor a given embodiment. The term permits the presence of one or moreadditional elements that do not materially affect the basic and novel orfunctional characteristic(s) of that (those) embodiment(s).

The term “consisting of” refers to articles, compositions, methods, andrespective components thereof as described herein that are exclusive ofany element not recited in that description of the embodiment.

The terms “e.g.,” “such as”, and like terms mean “for example”, and thusdo not limit the term or phrase they explain, whereas the term “i.e.,”and like terms mean “that is”, thus limiting the term or phrase itexplains.

As used herein, the term “epitope” or “epitopes,” or “epitopes ofinterest” refer to a site(s) on any molecule that is recognized and iscapable of binding to a complementary site(s) on its specific bindingpartner. The epitope-bearing molecule and specific binding partner arepart of a specific binding pair. For example, an epitope can be apolypeptide, protein, hapten, carbohydrate antigen (such as, but notlimited to, glycolipids, glycoproteins or lipopolysaccharides) orpolysaccharide and its specific binding partner, can be, but is notlimited to, an antibody, e.g., an autoantibody. Typically an epitope iscontained within a larger molecular framework (e.g., in the context ofan antigenic region of a protein, the epitope is the region or fragmentof the protein having the structure capable of being bound by anantibody reactive against that epitope) and refers to the preciseresidues known to contact the specific binding partner. As is known, itis possible for an antigen or antigenic fragment to contain more thanone epitope.

The term “graft” as used herein refers to biological material derivedfrom a donor for transplantation into a recipient. Grafts include suchdiverse material as, for example, isolated cells such as islet cells;tissue such as the amniotic membrane of a newborn, bone marrow,hematopoietic precursor cells, and ocular tissue, such as cornealtissue; and organs such as skin, heart, liver, spleen, pancreas, thyroidlobe, lung, kidney, tubular organs (e.g., intestine, blood vessels, oresophagus), etc. The tubular organs can be used to replace damagedportions of esophagus, blood vessels, or bile duct. The skin grafts canbe used not only for burns, but also as a dressing to damaged intestineor to close certain defects such as diaphragmatic hernia. The graft isderived from any mammalian source, including human, whether fromcadavers or living donors. Preferably the graft is bone marrow or anorgan such as heart and the donor of the graft and the host are matchedfor HLA class II antigens.

“Herein” means in the present application, including anything that maybe incorporated by reference.

As used herein, “specific” or “specificity” in the context of aninteraction between members of a specific binding pair (e.g., an antigenand antibody that specifically binds such antigen) refers to theselective reactivity of the interaction. The phrase “specifically bindsto” and analogous terms refer to the ability of antibodies tospecifically bind to (e.g., preferentially react with) an antigen andnot specifically bind to other entities. Antibodies or antigen-bindingantibody fragments that specifically bind to a particular antigen can beidentified, for example, by diagnostic immunoassays (e.g.,radioimmunoassays (“RIA”) and enzyme-linked immunosorbent assays(“ELISAs”), surface plasmon resonance, or other techniques known tothose of skill in the art. In one embodiment, the term “specificallybinds” or “specifically reactive” indicates that the binding preference(e.g., affinity) for the target analyte is at least about 2-fold, morepreferably at least about 5-fold, 10-fold, 100-fold, 1,000-fold, amillion-fold or more over a non-specific target molecule (e.g., arandomly generated molecule lacking the specifically recognizedsite(s)).

The term “labeled” refers to a molecule (e.g., an antibody,nanoparticle, etc.) that is labeled with a detectable label or becomeslabeled with a detectable label during use. A “detectable label”includes any moiety that is detectable or that can be rendereddetectable. With reference to a labeled separable particle, a “directlabel” is a detectable label that is attached to or associated with,covalently or non-covalently, the particle, and an “indirect label” is adetectable label that specifically binds the particle. Thus, an indirectlabel includes a moiety that is the specific binding partner of a moietyof the detection agent. Biotin and avidin are examples of such moietiesthat can be employed, for example, by contacting a biotinylated antibodywith labeled avidin to produce an indirectly labeled antibody (and thuslabeled nanomagnetic particle). A “label” refers to a detectablecompound or composition, such as one that is conjugated directly orindirectly to a target-specific binding member. The label may itself bedetectable by itself (e.g., a Raman label, a radioisotope, a fluorescentlabel, etc.) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition that isdetectable.

The term “magnetic particles” refers to ferromagnetic, superparamagnetic, or paramagnetic solid phases such as colloidal particles,microspheres, nanoparticles, or beads. The particles may be used insuspension or in a lyophilized state.

A “microparticle” refers to a small particle that is recoverable by anysuitable process, e.g., magnetic separation, buoyant separation,ultracentrifugation, etc. Microparticles typically have an averagediameter on the order of about 1 micron or less.

A “microbubble” refers to a small buoyant particle that is separated andrecoverable by any suitable process, e.g., floatation withoutcentrifugation, accelerated floatation with centrifugation, acceleratedfloatation with high buoyancy/low density buffers, removal and/ortransfer by pipetting, removal by pouring, removal by aspiration, etc.Microbubbles typically range in size from about 0.1 to about 100microns, typically about 1 to about 50 microns, and frequently about 2to about 20 or 30 microns in diameter. Microbubbles may be made of anysuitable material(s), e.g., glass, phospholipid, heated BSA, etc.Microbubbles may be filled with ambient air, a specific gas or mixtureof gases, or low density/high buoyancy fluid(s).

A “nanoparticle” refers to a small particle that is recoverable by anysuitable process, e.g., magnetic separation or association,ultracentrifugation, etc. Nanoparticles typically have an averagediameter on the order of about 500 nanometers (nm) or less, preferablyfrom about 20 nm to about 300 nm, or any size or size range within such1 nm-about 500 nm size range.

A “patentable” process, machine, or article of manufacture according tothe invention means that the subject matter satisfies all statutoryrequirements for patentability at the time the analysis is performed.For example, with regard to novelty, non-obviousness, or the like, iflater investigation reveals that one or more claims encompass one ormore embodiments that would negate novelty, non-obviousness, etc., theclaim(s), being limited by definition to “patentable” embodiments,specifically excludes the unpatentable embodiment(s). Also, the claimsappended hereto are to be interpreted both to provide the broadestreasonable scope, as well as to preserve their validity. Furthermore, ifone or more of the statutory requirements for patentability are amendedor if the standards change for assessing whether a particular statutoryrequirement for patentability is satisfied from the time thisapplication is filed or issues as a patent to a time the validity of oneor more of the appended claims is questioned, the claims are to beinterpreted in a way that (1) preserves their validity and (2) providesthe broadest reasonable interpretation under the circumstances.

A “plurality” means more than one.

The term “select”, when used in reference to a cell or population ofcells, refers to choosing, separating, segregating, and/or selectivelypropagating one or more cells having a desired characteristic.

The terms “separated”, “purified”, “isolated”, and the like mean thatone or more components of a sample or reaction mixture have beenphysically removed from, or diluted in the presence of, one or moreother components present in the mixture.

The term “species” is used herein in various contexts, e.g., aparticular target biomolecule species. In each context, the term refersto a population of chemically indistinct molecules of the sort referredin the particular context.

A “subject” (or “patient”) means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates, for example, include chimpanzees, cynomologousmonkeys, spider monkeys, and macaques, e.g., Rhesus monkeys. Rodentsinclude mice, rats, woodchucks, ferrets, rabbits and hamsters. Domesticand game animals include cows, horses, pigs, deer, bison, buffalo,feline species, e.g., domestic cat, canine species, e.g., dog, fox,wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout,catfish and salmon. Patient or subject includes any subset of theforegoing, e.g., all of the above. In certain embodiments, the subjectis a mammal, e.g., a primate, e.g., a human.

The term “transplant” and variations thereof refers to the insertion ofa graft into a host, whether the transplantation is syngeneic (where thedonor and recipient are genetically identical), allogeneic (where thedonor and recipient are of different genetic origins but of the samespecies), or xenogeneic (where the donor and recipient are fromdifferent species). Thus, in a typical scenario, the host is human andthe graft is an isograft, derived from a human of the same or differentgenetic origins. In another scenario, the graft is derived from aspecies different from that into which it is transplanted, such as ababoon heart transplanted into a human recipient host, and includinganimals from phylogenetically widely separated species, for example, apig heart valve, or animal beta islet cells or neuronal cellstransplanted into a human host.

The terms “treat,” “treatment,” “treating,” or “amelioration” when usedin reference to a disease, disorder or medical condition, refer totherapeutic treatments for a condition, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a symptom or condition. The term “treating”includes reducing or alleviating at least one adverse effect or symptomof a condition. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a condition is reduced or halted. Thatis, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation or at least slowing of progress orworsening of symptoms that would be expected in the absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of the deficit, stabilized (i.e., not worsening) state of health,delay or slowing of the disease progression, and amelioration orpalliation of symptoms. Treatment can also include the subject survivingbeyond when mortality would be expected statistically.

REFERENCES

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All of the compositions, articles, devices, systems, and methodsdisclosed and claimed herein can be made and executed without undueexperimentation in light of the present disclosure. While thecompositions, articles, devices, systems, and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions, articles, devices, systems, and methods withoutdeparting from the spirit and scope of the invention. All suchvariations and equivalents apparent to those skilled in the art, whethernow existing or later developed, are deemed to be within the spirit andscope of the invention as defined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications are herein incorporated by reference intheir entirety for all purposes and to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference in its entirety for any and all purposes.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims, which may also containeven further embodiments of the invention.

What is claimed is:
 1. A method of separating at least one targetbiomolecule species from a biological sample, comprising: (a) in areaction mixture, contacting a biological sample known or suspected tocontain first and second biomolecule species of interest with a targetedmagnetic particle species, optionally a targeted nanomagnetic particlespecies, that targets the first biomolecule species of interest to formfirst target biomolecule/magnetic particle complexes and a targetedbuoyant particle species, optionally a targeted buoyant microparticle,optionally a microbubble, species that targets the second biomoleculespecies of interest to form second target biomolecule/buoyant particlecomplexes; (b) using a magnetic field to isolate the firstbiomolecule/magnetic particle complexes from the reaction mixture;and/or, (c) using buoyancy/floatation properties to separate the secondtarget biomolecule/buoyant particle complexes from the reaction mixture.2. A method according to claim 1 wherein the targeted magnetic particlespecies is a targeted nanomagnetic particle species that comprises: (i)a magnetic core particle; (ii) a glass layer encapsulating the magneticcore particle; (iii) a protein/polymer composite layer bound to theglass layer; and (iv) a targeting moiety that targets the firstbiomolecule species of interest and comprises one member of abioaffinity ligand pair bound to the protein/polymer composite layer. 3.A method according to claim 2 wherein molecules of the targetednanomagnetic particle species have a diameter ranging from about 5 nm toabout 500 nm, preferably from about 30 nm to about 300 nm.
 4. A methodaccording to claim 2 wherein the magnetic core particles of the targetednanomagnetic particle species comprise magnetite (Fe₃O₄) crystals,optionally wherein the magnetite crystals have a diameter ranging fromabout 5 nm to about 300 nm.
 5. A method according to claim 2 wherein theglass layer of the targeted nanomagnetic particle species is a silanelayer formed from organofunctional alkoxysilane molecules, optionallyorganofunctional alkoxysilane molecules that comprise a couplable endgroup, optionally a couplable end group selected from the groupconsisting of an amino, sulphydryl, carboxyl, and hydroxyl end group. 6.A method according to claim 2 wherein the protein/polymer compositelayer of the targeted nanomagnetic particle species is covalently boundto the glass layer, optionally wherein the protein/polymer compositelayer is comprised of serum albumin, optionally bovine or human serumalbumin, dextran or casein and wherein optionally the protein/polymercomposite layer is permanently bound by heating the composition fromabout 45° C. to about 85° C.
 7. A method according to claim 2 whereinthe targeting moiety of the targeted nanomagnetic particle species isselected from the group consisting of an antibody, an antigen-bindingantibody fragment, a recombinant antibody, a cell surface receptor, aligand-binding extracellular domain of a cell surface receptor, anaptamer, a nucleic acid, avidin, streptavidin, and biotin.
 8. A methodaccording to claim 1 wherein the targeted magnetic particles and/or thetargeted buoyant particles each independently further comprise adetectable label.
 9. A method according to claim 2 wherein the magneticcore particles of the targeted nanomagnetic particle species comprise aferrous oxide, optionally Fe₃O₄ or Fe₂O₃; a chromium oxide, optionallyCrO₃; or a stable metal oxide that comprises a substituted metal ionselected from the group consisting of Mn, Co, Ni, Zn, Gd, and Dy.
 10. Amethod according to claim 1 wherein the targeted buoyant particlespecies comprises targeted buoyant microparticles, optionally targetedmicrobubbles, and a targeting moiety selected from the group consistingof an antibody, an antigen-binding antibody fragment, a recombinantantibody, a cell surface receptor, a ligand-binding extracellular domainof a cell surface receptor, an aptamer, a nucleic acid, avidin,streptavidin, and biotin.
 10. A method according to claim 1 wherein thefirst biomolecule species is a cell-surface antigen of a cell typeuseful for cell therapy, optionally human cell therapy.
 11. A methodaccording to claim 1 used to prepare an enriched cell population,wherein the cells of the enriched cell population express the firstbiomolecule species as a cell-surface antigen.
 12. An isolated, enrichedcell population, wherein the enriched cell population is produced usinga method according to claim
 11. 13. A method of administering anenriched cell population to a subject, comprising administering to asubject an isolated, enriched cell population according to claim 12,wherein the enriched cell population is enriched for stem cells thatexpress the first biomolecule species as a cell-surface antigen.
 14. Akit for performing a method according to claim 1, the kit comprising:(a) a composition that comprises a targeted magnetic particle species,optionally a targeted nanomagnetic particle species, that targets afirst biomolecule species of interest; (b) a composition that comprisesa targeted buoyant particle species, optionally a targeted buoyantmicroparticle species, optionally a targeted buoyant microbubblespecies, that targets a second biomolecule species of interest, whichcomposition targets a different biomolecule species as compared to thattargeted by the composition comprising the targeted magnetic particlespecies; and (c) instructions for performing a method according to claim1 using the targeted magnetic particle species and targeted buoyantparticle species.
 15. A kit according to claim 14 that comprises aplurality of targeted magnetic particle species, wherein the biomoleculespecies of interest targeted by each targeted magnetic particle speciesis different from other biomolecule species of interest targeted byother targeted magnetic particle species and the targeted buoyantmicroparticle species in the kit, wherein the different targetedmagnetic particle species are in the same or different compositions inthe kit.
 16. A kit according to claim 14 that comprises a plurality oftargeted buoyant particle species, wherein the biomolecule species ofinterest targeted by each targeted buoyant particle species is differentfrom other biomolecule species of interest targeted by other targetedmagnetic particle species and targeted buoyant particle species in thekit, wherein the different targeted buoyant particle species are in thesame or different compositions in the kit.